U.S. patent application number 16/165400 was filed with the patent office on 2019-04-04 for nucleic acid detection combining amplification with fragmentation.
This patent application is currently assigned to Quest Diagnostics Investments LLC. The applicant listed for this patent is Quest Diagnostics Investments LLC. Invention is credited to Richard A. Bender, Kevin Z. Qu, Heather R. Sanders, Charles M. Strom.
Application Number | 20190100808 16/165400 |
Document ID | / |
Family ID | 40850961 |
Filed Date | 2019-04-04 |
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United States Patent
Application |
20190100808 |
Kind Code |
A1 |
Sanders; Heather R. ; et
al. |
April 4, 2019 |
NUCLEIC ACID DETECTION COMBINING AMPLIFICATION WITH
FRAGMENTATION
Abstract
Provided herein are methods and compositions for detection of a
nucleic acid target in a sample. The methods and compositions use
primer directed amplification in conjunction with nucleic acid
fragmentation. The methods have high sensitivity even in the
presence of a large amount of non-target nucleic acid. Also
provided are oligonucleotides and kits useful in the method.
Exemplary nucleic acid targets are those with mutant gene sequence
such as mutant sequence of the EGFR, APC, TMPRSS2, ERG and ETV1
genes.
Inventors: |
Sanders; Heather R.; (RSM,
CA) ; Qu; Kevin Z.; (Lake Forest, CA) ; Strom;
Charles M.; (San Clemente, CA) ; Bender; Richard
A.; (Dana Point, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Quest Diagnostics Investments LLC |
Secaucus |
NJ |
US |
|
|
Assignee: |
Quest Diagnostics Investments
LLC
Secaucus
NJ
|
Family ID: |
40850961 |
Appl. No.: |
16/165400 |
Filed: |
October 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15712448 |
Sep 22, 2017 |
10106857 |
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16165400 |
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14679403 |
Apr 6, 2015 |
9783854 |
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15712448 |
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12035356 |
Feb 21, 2008 |
8999634 |
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14679403 |
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61007928 |
Jun 8, 2007 |
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60926611 |
Apr 27, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/6886 20130101; C12Q 2600/118 20130101; C12Q 2600/156
20130101; C12Q 2521/301 20130101; C12Q 2600/16 20130101; C12Q
1/6806 20130101; C12Q 2531/113 20130101 |
International
Class: |
C12Q 1/6886 20060101
C12Q001/6886; C12Q 1/6806 20060101 C12Q001/6806 |
Claims
1-5. (canceled)
6. A kit comprising one or more oligonucleotide primer pairs,
wherein at least one oligonucleotide of at least one primer pair
comprises a sequence selected from the group consisting of SEQ ID
NOs: 1-12, at least one oligonucleotide of the primer pair is
labeled with a detectable moiety.
7. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:1 and a reverse primer of SEQ
NO:2.
8. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:3 and a reverse primer of SEQ
NO:4.
9. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:5 and a reverse primer of SEQ
NO:6.
10. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:7 and a reverse primer of SEQ
NO:8.
11. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:9 and a reverse primer of SEQ
NO:10.
12. The kit of claim 6, wherein the oligonucleotide primer pair
comprises a forward primer of SEQ NO:11 and a reverse primer of SEQ
NO:12.
13. The kit of claim 6, wherein the detectable moiety is a
fluorescent dye.
14. The kit of claim 6, wherein the detectable moiety is a
fluorescent dye.
15. The kit of claim 6, wherein different pairs of primers in the
kit are labeled with different distinguishable detectable
moieties.
16. The kit of claim 6, wherein the at least one primer pair
comprises a forward primer and a reverse primer labeled with
different detectable moieties.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation of U.S.
application Ser. No. 15/712,448, filed Sep. 22, 2017, which is a
Divisional of U.S. application Ser. No. 14/679,403, filed Apr. 6,
2015, which is a Continuation of U.S. application Ser. No.
12/035,356, filed Feb. 21, 2008, which claims priority to U.S.
Provisional Patent Application No. 60/926,611, Titled: Nucleic Acid
Detection Combining Amplification With Fragmentation, filed Apr.
27, 2007 and U.S. Provisional Patent Application No. 61/007,928,
Titled: Nucleic Acid Detection Combining Amplification With
Fragmentation, filed Jun. 8, 2007, which are incorporated herein by
reference.
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-WEB and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 18, 2018, is named sequence.txt and is 17 KB.
FIELD OF THE INVENTION
[0003] Provided herein are methods and compositions for detecting
target nucleic acid such as mutant nucleic acid. The methods and
compositions combine amplification with nucleic acid fragmentation,
are useful for detecting very low amounts of target nucleic acids,
even in the presence of large amounts of non-target nucleic
acids.
BACKGROUND OF THE INVENTION
[0004] Although nucleic acid assays are known to offer a high
degree of specificity, there are limits in the sensitivity of such
assays, particularly when the target nucleic acid to be detected is
present in relatively low quantities compared to non-target nucleic
acid. In the case of cancer, the ability to detect the presence of
a small amount of a cancer specific mutant nucleic acid allows for
early cancer diagnosis and offers the possibility of more effective
therapeutic intervention. However, detection can be challenging if
the sample of nucleic acid being tested has very little of the
mutant nucleic acid and if there is an excess of normal nucleic
acid in the sample. Although a tumor biopsy may contain significant
mutant nucleic acids, a plasma sample from a cancer patient may
contain one or only a few copies of a mutant nucleic acid of
interest. Amplification methods such as PCR may detect a few copies
of a mutant nucleic acid, however, the abundance of normal nucleic
acid in samples such as plasma can interfere.
[0005] Focus has been placed on identifying tumor-derived mutations
in circulating DNA found in plasma or serum of solid tumor patients
as a noninvasive and early diagnostic tool. Confirmed reports of
the presence of solid tumor-derived mutations found in circulating
DNA include, but are not limited to, patients with colorectal
tumors, pancreatic cancer, breast cancer, head and neck squamous
cell carcinoma, and lung cancer (Hibi et al. 1998, Chen et al.
1999, Diehl et al. 2005, Coulet et al. 2000, Hagiwara 2006, Kimura
et al. 2006).
[0006] Reports have also demonstrated that cancer patients show
elevated levels of circulating DNA and have proposed use of DNA
quantification as prognostic and diagnostic factors (Gautschi et
al. 2004, Goebel et al. 2005, Sozzi et al. 2001, Herrera et al.
2005, Pathak et al. 2006). This has led to efforts to describe the
origin of such elevated levels of DNA. While still under
investigation, the sharp increase in circulating DNA is not likely
attributed to DNA released from tumor cells. In fact, analysis of
mutations present in the plasma of patients with colorectal tumors
revealed that the levels of mutations found in circulating DNA did
not increase proportionally with the overall elevated levels of
circulating DNA (Diehl et al. 2005). Thus while some cancer
patients show elevated levels of plasma DNA, detection of
tumor-derived mutations will require the ability to detect very few
mutations in the presence of larger amounts of wild type DNA.
[0007] A number of strategies have been described for detecting low
copy number nucleic acid targets. Methods including allele-specific
PCR of p53 and ABL kinase domain mutations have demonstrated
sensitivities ranging from 0.1-0.01% and in one mutation, 0.001%
(Righetti et al. 1999, Coulet et al. 2000, and Kang et al 2006).
Ohnishi, H., et al. reported a method of amplification using a
mutation specific primer that spans a deletion site and does not
anneal to the wild-type sequence. Ohnishi, H., et al., 15(2)
Diagnostic Molecular Pathology 101-108 (2006). Mutation specific
primers of the Scorpion type also have been reported. Kimura, H. et
al., 12(13) Clinical Cancer Research 3915-3921 (2006); Newton, C.
R., et al., 17(7) Nucleic Acids Research 2503-2516 (1989); and
Whitcombe, D. et al., 17 Nature Biotechnology 804-807 (1999)
(describing Scorpion ARMS primers and strategies for primer
design). Methods that enrich mutant nucleic acid by digesting
wild-type DNA with restriction enzymes prior to amplification have
been reported. Asano, H., et al., 12(1) Clinical Cancer Research
43-48 (2006); Gocke, C., et al., U.S. Pat. No. 6,630,301. The Asano
et al., method uses multiple PCR reactions. A first PCR reaction is
used to remove an upstream restriction enzyme recognition site.
Following the first PCR, a restriction digestion is performed.
After digestion, a second PCR reaction is used to amplify the
target sequence.
SUMMARY OF THE INVENTION
[0008] Provided herein are methods and compositions for detecting
target nucleic acids at very low levels and in the presence of
large amounts of non-target nucleic acids. Generally, a target and
non-target nucleic acid are distinguished by the presence or
absence of a fragmentation site, such as a restriction enzyme
recognition site. By differentiating the target and non-target by a
fragmentation site, the methods and compositions used herein can be
used with various nucleic acid detection methods known in the art,
such as PCR.
[0009] As used herein, the term "target" nucleic acid refers to a
nucleic acid which contains an allele or a mutant nucleic acid
sequence. A mutant nucleic acid sequence may be any mutant sequence
including but not limited to substitution, insertion, deletion, and
translocation.
[0010] As used herein, the term "non-target" or "other" nucleic
acid used in reference to a target nucleic acid means a nucleic
acid that does not contain the target sequence. For example, a
non-target nucleic acid of a target nucleic acid encoding an
allelic sequence encompasses nucleic acid that contains an
alternative allele. The non-target of a nucleic acid containing a
mutant sequence is a nucleic acid that contains normal or wild-type
nucleic acid sequence with respect to the mutant sequence.
[0011] As used herein, the term "locked nucleic acid" or "LNA"
refers to bicyclic nucleic acid analogs contain one or more 2'-O,
4'-C methylene linkage(s), which effectively locks the furanose
ring in a C3'-endo conformation. This methylene linkage restricts
the flexibility of the ribofuranose ring and locks the structure
into a rigid bicyclic formation. Because of its structural
conformation, locked nucleic acids demonstrate a much greater
affinity and specificity to their complementary nucleic acids than
do natural DNA counterparts and increases the thermal and chemical
stability of a primer/target nucleic acid duplex. LNAs will
hybridize to complementary nucleic acids even under adverse
conditions, such as under low salt concentrations and in the
presence of chaotropic agents. According to one aspect of the
invention, locked nucleic acids increase the melting point of the
primer/target nucleic acid duplex by about 3 to about 8.degree.
Celsius per locked nucleic acid base incorporated in the primer.
The basic structural and functional characteristics of LNAs and
related analogues are disclosed in various publications and
patents, including WO 99/14226, WO 00/56748, WO 00/66604, WO
98/39352, U.S. Pat. Nos. 6,043,060, and 6,268,490; see also,
Braasch et al., "Locked nucleic acid (LNA): fine-tuning the
recognition of DNA and RNA," Chem. Biol. 8(1):1
[0012] Locked nucleic acid bases may be interspersed throughout a
strand of a primer, placed consecutively or placed singularly in
predetermined locations. In one embodiment, the mutation specific
primer comprises a locked nucleic acid base at its 3' terminus. In
another embodiment, the mutation specific primer comprises a locked
nucleic acid base at its N-1 (i.e., penultimate) base. The mutant
base may be a locked nucleic acid.
[0013] In one aspect, provided herein is a method for detecting the
presence or absence of a target nucleic acid by testing a sample
that potentially contains the target nucleic acid in the presence
of non-target nucleic acid, the method includes: a) fragmenting the
sample nucleic acid under conditions such that a subsequent
amplification directed to the target nucleic acid results in an
increased detection of the target nucleic acid over the non-target
nucleic acid as compared to amplification without fragmentation; b)
amplifying the target nucleic acid with a pair of primers, where a
first primer is specific for the target nucleic acid; and c)
detecting the presence or absence of an amplification product,
which indicates the presence or absence of the target nucleic acid
in the sample.
[0014] In another aspect, provided herein is a method for
diagnosing a cancer or detecting the presence of a tumor cell by
determining if an individual has a mutant sequence associated with
the cancer or tumor cell type, the method includes: a) obtaining a
sample including nucleic acid from the individual; b) fragmenting
the sample nucleic acid under conditions such that a subsequent
amplification directed to the target nucleic acid results in an
increased detection of the target nucleic acid over the non-target
nucleic acid as compared to amplification without fragmentation; c)
amplifying the target nucleic acid with a pair of primers, where a
first primer is specific for the target nucleic acid; and d)
detecting the presence or absence of an amplification product
containing the mutant sequence, where diagnosis of cancer is
determined by the presence absence or amount of amplification
product containing the mutant sequence.
[0015] In yet another aspect, provided herein is a method for
determining prognosis with cancer by determining if an individual
has a mutant sequence associated with the cancer, the method
includes: a) obtaining a sample containing nucleic acid from the
individual; b) fragmenting the mutant nucleic acid under conditions
such that a subsequent amplification directed to the mutant nucleic
acid results in an increased detection of the mutant nucleic acid
over the non-mutant nucleic acid as compared to amplification
without fragmentation; c) amplifying the mutant nucleic acid with a
pair of primers, where a first primer is specific for the mutant
nucleic acid; and d) detecting the presence, absence and/or amount
of an amplification product containing the mutant sequence, where
the likelihood of an outcome in the individual is associated with
the presence and or amount of mutant nucleic acid sequence.
[0016] In still yet another aspect, provided herein is a method for
determining drug sensitivity of an individual diagnosed with
cancer, the method includes: a) obtaining a sample comprising
nucleic acid from the individual; b) fragmenting the mutant nucleic
acid under conditions such that a subsequent amplification directed
to the mutant nucleic acid results in an increased detection of the
mutant nucleic acid over the non-mutant nucleic acid as compared to
amplification without fragmentation; c) amplifying the mutant
nucleic acid with a pair of primers, where a first primer is
specific for the mutant nucleic acid; d) detecting the presence,
absence and/or amount of an amplification product containing the
mutant sequence; and e) relating the presence, absence and/or
amount of an amplification product containing the mutant sequence
to cancer drug sensitivity. Some examples of mutations that affect
drug sensitivity which may be targeted by the assay methods
described herein are described in Lynch, et al., 350(21) NEJM
2129-2139 (2004); Bell, et al., US Patent App. No. 20060147959
(2005) (determining tyrosine kinase inhibitor, i.e., gefitinib and
erlotinib sensitivity by detecting EGFR mutations); and Sawyers, et
al., U.S. Patent Appl. No. 2006/0269956 (describing mutations that
affect drug resistance to BCR-ABL kinase activity inhibitors
typically used to treat CML due to the T315I mutation in the Abl
gene).
[0017] In certain embodiments of the aspects provided herein, the
mutated nucleic acid sequence is due to a deletion, insertion,
substitution and/or translocation or combinations thereof. In
preferred embodiments, fragmentation of nucleic acid sequence in
which cleavage of wild-type sequence is with a restriction enzyme.
Such pre-amplification digestion treatment allows for fragmentation
to destroy or substantially decrease the number of wild-type
sequences that might be amplified. In yet more preferred
embodiments, the fragmentation using a restriction enzyme is
combined with the use of a mutation specific primer (or mutated
sequence primer).
[0018] In preferred embodiments, a mutated sequence destroys or
disrupts a restriction enzyme recognition site present in the
corresponding wild-type sequence and that a mutation specific
primer can be designed to bind to the mutated version of the
sequence and not its wild-type counterpart. For example a mutation
specific primer can overlap a border region, which is a region that
contains portions of both a wild-type sequence adjacent to a
portion of the mutated sequence. In further examples, if a mutation
is the result of a deletion, such as the 15 bp deletion in exon 19
of the Epidermal Growth Factor Receptor (EGFR) gene (E746_A750del),
a mutation specific primer could be designed, as illustrated in
FIG. 1, so as to span a new site in the DNA which arises from the
deletion. Other methods of detecting EGFR nucleic acid are
described in U.S. Pat. No. 6,759,217 (which describes detecting
EGFR nucleic acid in plasma or serum), U.S. Pat. Nos. 6,127,126 and
5,981,725 (both disclose detecting nucleic acid encoding an EGFR
mutant protein type II for a mutation in which a portion of the
extracellular domain of EGFR is deleted). If a mutation is due to
an insertion, a mutation specific primer could be designed to span
either or both junctions where the inserted sequence is adjacent to
wild-type sequence. If a mutation is due to one or more
substitutions, then a mutation specific primer could be designed to
span any or all of the substitutions. If a mutation is due to a
translocation, then a mutation specific primer could be designed to
span one or both junctions of the translocated sequence, in any
region where the sequence is altered by the translocation. These
examples are merely exemplary and provide guidance to one of skill
in the art to design various permutations of primers that would
anneal to a mutated sequence and not a wild-type sequence which are
appropriate for the methods and compositions provided herein.
[0019] In one approach, a sample is assayed for the presence or
absence of a mutated sequence by amplification and detection of the
resulting amplification products. In a preferred embodiment,
amplification of target nucleic acids is accomplished by polymerase
chain reaction (PCR).
[0020] Single or multiple mutant sequences can be assayed.
Amplification of multiple mutant sequences can be performed
simultaneously in a single reaction vessel, e.g., multiplex PCR. In
this case, probes may be distinguishably labeled and/or amplicons
may be distinguishable by size differentiation. Alternatively, the
assay could be performed in parallel in separate reaction vessels.
In such later case, the probes could have the same label.
[0021] In certain embodiments of the aspects provided herein, the
methods further comprise a nucleic acid extraction step. Various
extraction nucleic acid methods are known in the art which can be
employed with the methods and compositions provided herein such as
lysis methods (such as alkaline lysis), phenol:chloroform and
isopropanol precipitation. Nucleic acid extraction kits can also be
used. Preferably, the extraction method is Agencourt Genfind.TM.,
Roche Cobas.RTM. or phenol:chloroform extraction using Eppendorf
Phase Lock Gels.RTM.. More preferably the nucleic acid is extracted
using Agencourt Genfind.TM.
[0022] Also provided are exemplary oligonucleotides useful in the
methods and kits described herein.
[0023] The following mutated sequences can be detected with the
methods and compositions provided herein. The methods outlined for
detection of specific deletion mutations, insertion mutations,
point mutations and fusion transcripts can be applied to any
biomarker which may be used with fragmentation, particularly when a
restriction digestion recognition site is disrupted. Embodiments of
specific primer designs are described below but the sequence will
vary to fit the mutation to be detected. Restriction enzyme
digestion sites will also depend on the sequence of the wild-type
sequence as compared to the mutated nucleic acid or fusion
transcript. The frequency of various restriction sites found in DNA
virtually ensures that a site unique to the wild-type DNA of
interest can be found for any mutation detection assay, thus this
methodology is applicable to a wide array of cancer biomarkers.
[0024] In one approach, a mutation specific primer is designed for
detecting a deletion mutation. Mutation specific primer can be
designed to span the deleted region such that the primer contains
wild-type sequence that lies 5' and 3' of the deleted region or the
complement thereof. Thus, the mutation specific primer cannot bind
to the wild-type sequence and cannot produce an amplicon.
[0025] In one approach, a mutation specific primer is designed for
detecting an insertion mutation. A mutation specific primer can be
designed to span all or a portion of the inserted region such that
the primer includes all or a part of the inserted region. A primer
could be designed to span the either or both junctions of the
inserted sequence, for example, the primer sequence would include a
portion of wild-type sequence that is adjacent to the inserted
sequence or the complement thereof. Thus, the mutation specific
primer is not complementary to the wild-type sequence and cannot
produce an amplicon.
[0026] In one approach, a mutation specific primer is designed for
detecting one or more substitution mutations. A mutation specific
primer can be designed to include one or more substitutions or the
complement thereof. For example, the 3' nucleotide of the primer
can be designed such that it contains the mutated base pair and
does not bind hybridize, or base pair, in the wild-type gene and
thus cannot elongate.
[0027] In one approach, a mutation specific primer is designed for
detecting one or more translocation mutations. A mutation specific
primer can be designed to span the junction of the translocation or
the complement thereof. A primer pair could be designed to so that
one primer is upstream of the translocation junction and the second
is downstream of the junction. Thus, when the primer pair is used
on wild-type sequence, no amplification products will be produced
because the locations of the primers relative to each other are
cannot be amplified. However, when the translocation is present,
the primers are in close enough proximity of each other such that
an amplification product can be produced. For example, the primer
can be designed to include a portion of the first gene and a
portion of the second gene, where the genes are located on
different chromosomes in wild-type form but are adjacent to one
another in the mutated form.
[0028] In certain embodiments, at least one primer of each primer
pair in the amplification reaction is labeled with a detectable
moiety. Thus, following amplification, the various target segments
can be identified by size and color. The detectable moiety is
preferably a fluorescent dye. In some embodiments, different pairs
of primers in a multiplex PCR may be labeled with different
distinguishable detectable moieties. Thus, for example, HEX and FAM
fluorescent dyes may be present on different primers in multiplex
PCR and associated with the resulting amplicons. In other
embodiments, the forward primer is be labeled with one detectable
moiety, while the reverse primer is labeled with a different
detectable moiety, e.g. FAM dye for a forward primer and HEX dye
for a reverse primer. Use of different detectable moieties is
useful for discriminating between amplified products which are of
the same length or are very similar in length. Thus, in certain
embodiments, at least two different fluorescent dyes are used to
label different primers used in a single amplification. In still
another embodiment, control primers can be labeled with one moiety,
while the patient (or test sample) primers can be labeled with a
different moiety, to allow for mixing of both samples (post PCR)
and the simultaneous detection and comparison of signals of normal
and test sample. In a modification of this embodiment, the primers
used for control samples and patient samples can be switched to
allow for further confirmation of results.
[0029] Analysis of amplified products from amplification reactions,
such as multiplex PCR, can be performed using an automated DNA
analyzer such as an automated DNA sequencer (e.g., ABI PRISM 3100
Genetic Analyzer) which can evaluate the amplified products based
on size (determined by electrophoretic mobility) and/or respective
fluorescent label.
[0030] The methods and compositions provided herein provide
increased sensitivity for detection of a mutated nucleic acid.
Preferably the methods can detect mutated nucleic acid that is
present in 10% or less, 1% or less, 0.1% or less, 0.01% or less,
0.001% or less, 0.0005% or less, 0.0003% or less, or 0.0002% or
less than the total nucleic acid of a sample.
[0031] Various other cancer biomarkers suitable for detection using
the methods and compositions provided herein include, but are not
limited to, breast cancer markers, such as, GSTP1, RASSF1A (both
described in Papadopoulou, E. et al., 1075 Ann. N.Y. Acad. Sci.
235-243 (2006); and RASSF1A (Papadopoulou, E., et al., and Coyle,
et al., 16(2) Cancer Epidemiol. Biomarkers Prev. 192-196 (2007)),
ATM (Papadopoulou, E., et al.), APC (Coyle, et al.), RARbeta2
(Hogue, M O, et al., 24(26) J. Clin. Oncol. 4262-4269, Epub 2006
Aug. 14 (2006)), and TP53 (Silva, J. M., et al., 8(12) Clin. Cancer
Res. 3761-3766 (2002)); ovarian cancer markers, such as p53
(Swisher, E. M., et al., 193(3) American Journal of Obstetrics and
Gynecology 662-667 (2005)); hepatocellular carcinoma markers, such
as, p53 mutations (Huang, X. H., et al., 9(4) World J
Gastroenterol. 692-695 (2003)), and p16 (Le Roux, E., 53(3) Rev.
Epidemiol. Sante Publique. 257-266 (2005)); and pancreatic cancer
markers, such as K-ras (Castells, A., et al., 17(2) J. Clin. Oncol.
578-584 (1999)).
[0032] Oligonucleotides or combinations of oligonucleotides that
are useful as primers or probes in the methods are also provided.
These oligonucleotides are provided as substantially purified
material.
[0033] Kits comprising oligonucleotides which may be primers for
performing amplifications as described herein also are provided.
Kits may further include oligonucleotides that may be used as
probes to detect amplified nucleic acid. Kits may also include
restriction enzymes for digesting non-target nucleic acid to
increase detection of target nucleic acid by the oligonucleotide
primers.
[0034] As used herein, the term "junction" refers to the position
where target and non-target sequences are adjacent to one another
due to a sequence change. For example, in the event of a
translocation between sequence 1, ATGC and sequence 2, CGTA, the
resulting mutated sequence or fusion sequence would be ATGCCGTA.
The junction in this translocation example would be "CC." For
example, in the even of an insertion of sequence 3, CCCC into
sequence 4, ATGC, the resulting mutated sequence would be ATCCCCGC.
The junctions in this event would be "TC" and "CG."
[0035] "Fragmentation" as used herein refers a process in which
longer lengths of nucleic acid are broken up into shorter lengths
of nucleic acid. Nucleic acids may be broken up or fragmented by
chemical or biochemical means, preferably nucleic acids are
fragmented in a manner that is reproducible, preferably nucleic
acids are fragmented by one or more restriction endonucleases. The
length of a fragment containing the nucleic acid segment of
interest can depend on the length of the nucleic acid segment of
interest as well as the restriction enzyme chosen to fragment the
DNA.
[0036] A "restriction endonuclease" or "restriction enzyme" as used
herein refers to an enzyme that cuts double-stranded DNA at a
specific sequence (i.e., the recognition sequence or site). The
frequency with which a given restriction endonuclease cuts DNA
depends on the length of the recognition site of the enzyme. For
example, some enzymes recognize sites that are four nucleotides
long (referred to as "four cutters"). In general one can estimate
how frequently an enzyme should cut a piece of DNA based the length
of the recognition site and the assumption that the probability of
any one nucleotide occurring at a given location is 1/4. In the
case of a "four cutter" a specific sequence of four nucleotides
must be present. Assuming that each nucleotide has an equal chance
(i.e., 1/4) of occurring at any particular site within the four
nucleotide sequence, then a four-cutter should on average cut once
every 256 base pairs (i.e., 1/4.times.1/4.times.1/4.times.1/4=
1/256). A similar calculation can be applied to any restriction
enzyme as long as the length of its recognition site is known,
making it possible to predict the size and number of a DNA
fragments that would be obtained by cutting a DNA molecule of known
size. This allows one of skill in the art to produce DNA fragments
of known size. Restriction endonucleases are obtained from bacteria
or are produced through recombinant technology and are readily
available through numerous commercial sources.
[0037] As used herein, the term "increased detection" refers to the
ability to detect lower amounts of target nucleic acid in the
presence of non-target nucleic acid. For example, as non-target
nucleic acid increases, fragmentation of the non-target nucleic
acid increases the ability to detect a smaller fraction of target
nucleic acid in total nucleic acid.
[0038] As used herein, the term "sample" or "test sample" refers to
any liquid or solid (or both) material can be used to test for the
presence of nucleic acids. In preferred embodiments, a test sample
is obtained from a biological source (i.e., a "biological sample"),
such as cells in culture or tissue cells from an animal,
preferably, a human. Preferred sample sources include, but are not
limited to, sputum (processed or unprocessed), bronchial alveolar
lavage (BAL), bronchial wash (BW), blood, bone marrow, bodily
fluids, cerebrospinal fluid (CSF), urine, plasma, serum or tissue
(e.g., biopsy material). A body fluid sample refers to fluid
containing samples from an individual including sputum (processed
or unprocessed), bronchial alveolar lavage (BAL), bronchial wash
(BW), blood, plasma, serum, and cerebrospinal fluid (CSF). The term
"patient sample" as used herein refers to a sample obtained from a
human seeking diagnosis and/or treatment of a disease.
[0039] As used herein, the term "oligonucleotide" refers to a short
polymer composed of deoxyribonucleotides, ribonucleotides or any
combination thereof. Oligonucleotides are generally between about
10, 11, 12, 13, 14 or 15 to about 150 nucleotides (nt) in length,
more preferably about 10, 11, 12, 13, 14 or 15 to about 150 nt,
more preferably about 10, 11, 12, 13, 14, or 15 to about 70 nt, and
most preferably between about 20 to about 26 nt in length. The
single letter code for nucleotides is as described in the U.S.
Patent Office Manual of Patent Examining Procedure, section 2422,
table 1. In this regard, the nucleotide designation "R" means
guanine or adenine, "Y" means thymine (uracil if RNA) or cytosine;
and "M" means adenine or cytosine. An oligonucleotide may be used
as a primer or as a probe.
[0040] As used herein, the term "detecting" used in context of
detecting a signal from a detectable label to indicate the presence
of a target nucleic acid in the sample does not require the method
to provide 100% sensitivity and/or 100% specificity. As is well
known, "sensitivity" is the probability that a test is positive,
given that the person has a target nucleic acid sequence, while
"specificity" is the probability that a test is negative, given
that the person does not have the target nucleic acid sequence. A
sensitivity of at least 50% is preferred, although sensitivities of
at least 60%, at least 70%, at least 80%, at least 90% and at least
99% are clearly more preferred. A specificity of at least 50% is
preferred, although sensitivities of at least 60%, at least 70%, at
least 80%, at least 90% and at least 99% are clearly more
preferred. Detecting also encompasses assays with false positives
and false negatives. False negative rates may be 1%, 5%, 10%, 15%,
20% or even higher. False positive rates may be 1%, 5%, 10%, 15%,
20% or even higher.
[0041] As used herein, the term "substantially purified" in
reference to oligonucleotides does not require absolute purity.
Instead, it represents an indication that the specified
oligonucleotide is relatively more pure than it is in the natural
environment. Such oligonucleotides may be obtained by a number of
methods including, for example, laboratory synthesis, restriction
enzyme digestion or PCR. A "substantially purified" oligonucleotide
is preferably greater than 50% pure, more preferably at least 75%
pure, and most preferably at least 95% pure.
[0042] As used herein, an oligonucleotide is "specific" for a
nucleic acid if the oligonucleotide has at least 50% sequence
identity with a portion of the nucleic acid when the
oligonucleotide and the nucleic acid are aligned. An
oligonucleotide that is specific for a nucleic acid is one that,
under the appropriate hybridization or washing conditions, is
capable of hybridizing to the target of interest and not
substantially hybridizing to nucleic acids which are not of
interest. Higher levels of sequence identity are preferred and
include at least 75%, at least 80%, at least 85%, at least 90%, at
least 95% and more preferably at least 98% sequence identity.
Sequence identity can be determined using a commercially available
computer program with a default setting that employs algorithms
well know in the art.
[0043] As used herein, the term "hybridize" or "specifically
hybridize" refers to a process where two complementary nucleic acid
strands anneal to each other under appropriately stringent
conditions. Hybridizations are typically and preferably conducted
with oligonucleotides Nucleic acid hybridization techniques are
well known in the art. See, e.g., Sambrook, et al., 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Press, Plainview, N.Y. Those skilled in the art understand how to
estimate and adjust the stringency of hybridization conditions such
that sequences having at least a desired level of complementarity
will stably hybridize, while those having lower complementarity
will not. For examples of hybridization conditions and parameters,
see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.;
Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology.
John Wiley & Sons, Secaucus, N.J.
[0044] The terms "target nucleic acid" or "target sequence" as used
herein refer to a sequence which includes an allele or mutation of
interest to be amplified and detected. Copies of the target
sequence which are generated during the amplification reaction are
referred to as amplification products, amplimers, or amplicons.
Target nucleic acid may be composed of segments of a chromosome, a
complete gene with or without intergenic sequence, segments or
portions of a gene with or without intergenic sequence, or sequence
of nucleic acids which probes or primers are designed. Target
nucleic acids may include a wild-type sequences, a mutation,
deletion or duplication, tandem repeat regions, a gene of interest,
a region of a gene of interest or any upstream or downstream region
thereof. Target nucleic acids may represent alternative sequences
or alleles of a particular gene. Target nucleic acids may be
derived from genomic DNA, cDNA, or RNA. As used herein target
nucleic acid may be DNA or RNA extracted from a cell or a nucleic
acid copied or amplified therefrom.
[0045] "Genomic nucleic acid" or "genomic DNA" refers to some or
all of the DNA from a chromosome. Genomic DNA may be intact or
fragmented (e.g., digested with restriction endonucleases by
methods known in the art). In some embodiments, genomic DNA may
include sequence from all or a portion of a single gene or from
multiple genes. In contrast, the term "total genomic nucleic acid"
is used herein to refer to the full complement of DNA contained in
the genome. Methods of purifying DNA and/or RNA from a variety of
samples are well-known in the art.
[0046] The term "flanking" as used herein means that a primer
hybridizes to a target nucleic acid adjoining a region of interest
sought to be amplified on the target. The skilled artisan will
understand that preferred primers are pairs of primers that
hybridize 3' from a region of interest, one on each strand of a
target double stranded DNA molecule, such that nucleotides may be
add to the 3' end of the primer by a suitable DNA polymerase.
[0047] The term "complement" "complementary" or "complementarity"
as used herein with reference to polynucleotides (i.e., a sequence
of nucleotides such as an oligonucleotide or a target nucleic acid)
refers to standard Watson/Crick pairing rules. The complement of a
nucleic acid sequence such that the 5' end of one sequence is
paired with the 3' end of the other, is in "antiparallel
association." For example, the sequence "5'-A-G-T-3'" is
complementary to the sequence "3'-T-C-A-5'." Certain bases not
commonly found in natural nucleic acids may be included in the
nucleic acids described herein; these include, for example,
inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), and Peptide
Nucleic Acids (PNA). Complementary need not be perfect; stable
duplexes may contain mismatched base pairs, degenerative, or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs. A complement sequence can also be a sequence of RNA
complementary to the DNA sequence or its complement sequence, and
can also be a cDNA.
[0048] The term "substantially complementary" as used herein means
that two sequences hybridize under stringent hybridization
conditions. The skilled artisan will understand that substantially
complementary sequences need not hybridize along their entire
length. In particular, substantially complementary sequences can
comprise a contiguous sequence of bases that do not hybridize to a
target sequence, positioned 3' or 5' to a contiguous sequence of
bases that hybridize under stringent hybridization conditions to a
target sequence.
[0049] The term "coding sequence" as used herein means a sequence
of a nucleic acid or its complement, or a part thereof, that can be
transcribed and/or translated to produce the mRNA for and/or the
polypeptide or a fragment thereof. Coding sequences include exons
in a genomic DNA or immature primary RNA transcripts, which are
joined together by the cell's biochemical machinery to provide a
mature mRNA. The anti-sense strand is the complement of such a
nucleic acid, and the encoding sequence can be deduced from
there.
[0050] The terms "amplification" or "amplify" as used herein
includes methods for copying a target nucleic acid, thereby
increasing the number of copies of a selected nucleic acid
sequence. Amplification may be exponential or linear. A target
nucleic acid may be either DNA or RNA. The sequences amplified in
this manner form an "amplicon." While the exemplary methods
described hereinafter relate to amplification using the polymerase
chain reaction (PCR), numerous other methods are known in the art
for amplification of nucleic acids (e.g., isothermal methods,
rolling circle methods, etc.). The skilled artisan will understand
that these other methods may be used either in place of, or
together with, PCR methods. See, e.g., Saiki, "Amplification of
Genomic DNA" in PCR Protocols, Innis et al., Eds., Academic Press,
San Diego, Calif. 1990, pp 13-20; Wharam, et al., Nucleic Acids
Res. 2001 Jun. 1; 29(11):E54-E54; Hafner, et al., Biotechniques
2001 April; 30(4):852-6, 858, 860 passim; Zhong, et al.,
Biotechniques 2001 April; 30(4):852-6, 858, 860.
[0051] The term "multiplex PCR" as used herein refers to
simultaneous amplification of two or more products which are each
primed using a distinct primer pair.
[0052] As used herein, a "primer" for amplification is an
oligonucleotide that specifically anneals to a target nucleotide
sequence and leads to addition of nucleotides to the 3' end of the
primer in the presence of a DNA or RNA polymerase. The 3'
nucleotide of the primer should generally be identical to the
target sequence at a corresponding nucleotide position for optimal
expression and amplification. The term "primer" as used herein
includes all forms of primers that may be synthesized including
peptide nucleic acid primers, locked nucleic acid primers,
phosphorothioate modified primers, labeled primers, and the
like.
[0053] "Sense strand" means the strand of double-stranded DNA
(dsDNA) that includes at least a portion of a coding sequence of a
functional protein. "Anti-sense strand" means the strand of dsDNA
that is the reverse complement of the sense strand.
[0054] As used herein, a "forward primer" is a primer that anneals
to the anti-sense strand of dsDNA. A "reverse primer" anneals to
the sense-strand of dsDNA.
[0055] As used herein, sequences that have "high sequence identity"
have identical nucleotides at least at about 50% of aligned
nucleotide positions, preferably at least at about 58% of aligned
nucleotide positions, and more preferably at least at about 76% of
aligned nucleotide positions.
[0056] As used herein "TaqMan.RTM. PCR detection system" refers to
a method for real time PCR. In this method, a TaqMan.RTM. probe
which hybridizes to the nucleic acid segment amplified is included
in the PCR reaction mix. The TaqMan.RTM. probe comprises a donor
and a quencher fluorophore on either end of the probe and in close
enough proximity to each other so that the fluorescence of the
donor is taken up by the quencher. However, when the probe
hybridizes to the amplified segment, the 5'-exonuclease activity of
the Taq polymerase cleaves the probe thereby allowing the donor
fluorophore to emit fluorescence which can be detected.
[0057] As used herein, "about" means plus or minus 10%.
BRIEF DESCRIPTION OF THE FIGURES
[0058] FIG. 1. Schematic diagram of primer placement for mutant
specific PCR of a deletion mutation exemplified by E746_A750del in
the EGFR gene. The deleted sequence is shown as a dashed line in
the EGFR wild-type DNA. Horizontal arrows indicate primer placement
for forward and reverse primers.
[0059] FIG. 2A. Nucleotide sequence of a portion of the sequence
coding for the wild-type form of the EGFR (SEQ ID NO:22). The two
segments of highlighted unbolded text together represent the
sequence for a forward mutation specific PCR primer (SEQ ID NO:1)
specific for the E746_A750del mutant EGFR gene. Only a portion of
the forward mutation specific primer is complementary to a
contiguous segment of the wild-type EGFR gene. Highlighted bolded
text indicates sequence for a reverse PCR primer (SEQ ID NO:2). The
15 bp region that is deleted is located between the two portions of
the forward primer in the E746_A750del mutant EGFR gene. Boxed TTAA
regions indicate MseI restriction sites.
[0060] FIG. 2B. Nucleotide sequence of a portion of the coding
region of the E746_A750del mutant of EGFR (SEQ ID NO:23.
Highlighted unbolded text indicates sequence of a forward mutation
specific PCR primer (SEQ ID NO:1) specific for the E746_A750del
mutant EGFR gene. Highlighted bolded text indicates sequence for a
reverse PCR primer (SEQ ID NO:2). Boxed TTAA regions indicate MseI
restriction sites.
[0061] FIG. 3. Schematic diagram of primer placement for mutant
specific PCR of an insertion mutation exemplified by the exon 16
mutation in the APC gene. Horizontal arrows indicate primer
placement for forward and reverse primers. The gray region
represents the inserted sequence. White stars indicate MnlI
restriction sites.
[0062] FIG. 4. Nucleotide sequence of a portion of APC gene showing
the exon 16 insertion sequence in unbolded text (SEQ ID NO:24).
Highlighted bolded text indicates the sequence for a forward PCR
primer (SEQ ID NO:3) and highlighted unbolded text indicates the
sequence of a reverse PCR primer (SEQ ID NO:4). Boxed GAGG and CCTC
regions indicate MnlI restriction sites.
[0063] FIG. 5. Schematic diagram of alternative primer placement
for mutant specific PCR of an insertion mutation exemplified by the
exon 16 mutation in the APC gene. Horizontal arrows indicate primer
placement for forward and reverse primers. The gray region
represents the inserted sequence. White stars indicate MnlI
restriction sites.
[0064] FIG. 6. Nucleotide sequence of a portion of APC gene showing
the exon 16 insertion sequence in unbolded text (SEQ ID NO:25).
Highlighted bolded text indicates the sequence for a forward PCR
primer (SEQ ID NO:5). Highlighted unbolded text indicates the
sequence of a reverse PCR primer (SEQ ID NO:6). Boxed GAGG and CCTC
regions indicate MnlI restriction sites.
[0065] FIG. 7. Schematic diagram of primer placement for mutant
specific PCR of a substitution mutation exemplified by point
mutation L858R in the EGFR gene. Horizontal arrows indicate primer
placement for forward and reverse primers. The boxed sequence
indicates the EaeI restriction site. The bolded "G" base pair
represents the substituted base (SEQ ID NO:26 and SEQ ID NO:27,
respectively in order of appearance).
[0066] FIG. 8. Nucleotide sequences of portions of wild-type (SEQ
ID NO:28) and mutant EGFR genes (SEQ ID NO:29). Highlighted
unbolded text indicates the sequence for a forward mutation
specific PCR primer (SEQ ID NO:7) specific for the L858R mutant
EGFR gene. Only a portion of the forward mutation specific primer
is complementary to a contiguous segment of the wild-type EGFR
gene. Highlighted bolded text indicates the sequence for a reverse
PCR primer (SEQ ID NO:8). The bolded boxed base pair "T" indicates
where the point mutation occurs in the L858R mutant EGFR gene. The
bolded "G" in the forward mutation specific primer is the location
of the locked nucleic acid. Highlighted unbolded text in the
wild-type EGFR gene sequence indicates where the forward mutation
specific primer would hybridize, or base pair. Boxed TTAA regions
indicate MseI restriction sites. Boxed YGGCCR region, where Y=C or
T; and R=A or G, indicates an EaeI restriction site.
[0067] FIG. 9. Schematic diagram of primer placement for mutant
specific PCR of a translocation mutation exemplified by the
TMPRSS2:ERG fusion transcript. Horizontal arrows indicate primer
placement for forward and reverse primers. White stars indicate
FatI restriction sites.
[0068] FIG. 10. Nucleotide sequences of portions of wild-type
TMPRSS2 (SEQ ID NO:30) and ERG (SEQ ID NO:31) and mutant fusion
(SEQ ID NO:32) gene, TMPRSS2:ERG. Highlighted bolded text indicates
the sequence for a forward mutation specific PCR primer (SEQ ID
NO:9). Highlighted unbolded text indicates the sequence for a
reverse mutation specific PCR primer (SEQ ID NO:10). Underlined
regions in the wild-type sequences correspond to the depicted
portion of the resulting fusion gene. Boxed CATG regions indicate
FatI restriction sites.
[0069] FIG. 11. Schematic diagram of primer placement for mutant
specific PCR of a translocation mutation exemplified by the
TMPRSS2:ETV1 fusion transcript. Horizontal arrows indicate primer
placement for forward and reverse primers. White stars indicate
FatI and HpyCH4V restriction sites.
[0070] FIG. 12. Nucleotide sequences of portions of wild-type
TMPRSS2 (SEQ ID NO:33) and ETVI (SEQ ID NO:34) and mutant fusion
(SEQ ID NO:35) gene, TMPRSS2:ETV1. Highlighted bolded text
indicates sequence for a forward mutation specific PCR primer (SEQ
ID NO:11). Highlighted unbolded text indicates sequence for a
reverse mutation specific PCR primer (SEQ ID NO:12). Underlined
regions in the wild-type sequences correspond to the depicted
portion of the resulting fusion gene. Boxed CATG and TGCA regions
indicate HpyCH4V restriction sites.
[0071] FIG. 13. Graphical depiction of results of sensitivity
comparison assay between Sanders method and Asano method for
detection of the E746_A750del mutation in EGFR.
[0072] FIG. 14. Comparison of eight DNA extraction methods. Columns
indicate the mean percent recovery of six plasma samples as
described in Example 5.
[0073] FIG. 15. Evaluation of detection between Agencourt
Genfind.TM., Roche Cobas.RTM. and phenol:chloroform extraction
using Eppendorf Phase Lock Gels.RTM. methods using spiked plasma
samples as described in Example 5. Columns represent the mean peak
intensity of E746_A750del PCR product from the six samples tested
for each method obtained from an ABI 3100 Genetic Analyzer.
[0074] FIG. 16. Evaluation of nucleic acid spiking conditions as
described in Example 5. Six nucleic acid carrier conditions include
1) no carrier, 2) no carrier plus 100 ng of normal DNA, 3) 1 .mu.g
of RNA carrier, 4) 1 .mu.g of RNA carrier plus 100 ng of normal
DNA, 5) 395 ng of normal DNA as a carrier, and 6) 778 ng of normal
DNA as a carrier. Columns represent the mean peak intensity of
E746_A750del PCR product.
DETAILED DESCRIPTION OF THE INVENTION
[0075] In accordance with the present invention, there are provided
methods for determining whether a sample contains target nucleic
acid. The methods outlined for detection of specific deletion
mutations, insertion mutations, point mutations and translocation
mutations can be applied to any biomarker in proximity to a
restriction digestion recognition site, preferably a restriction
digestion recognition site is disrupted by one or more mutations. A
blueprint of the primer designs is depicted below but primer
sequences will vary to fit the mutation to be detected. Restriction
enzyme digestion sites will also depend on the sequence of the
non-target sequence as compared to the target nucleic acid or
fusion transcript but can follow the formats below. The frequency
of various restriction sites found in DNA virtually ensures that a
site unique to the non-target DNA of interest can be found for any
target detection assay, thus these methodologies are applicable to
a wide array of cancer biomarkers.
Primers
[0076] For the methods provided herein, a single primer could be
used for detection, for example as in single nucleotide primer
extension, or a second primer can be used which can be upstream or
downstream of the mutation specific primer. One or more of the
primers used may be mutation specific primers. Preferably, the
mutation specific primer contains wild-type sequence, more
preferably at least about 3-40 consecutive nucleotides of wild-type
sequence.
Fragmentation
[0077] Fragmentation is preferably achieved by restriction enzyme
treatment or one of other methods of fragmentation well known in
the art. In order to reduce the likelihood of mis-priming or
inability of the decreased ability for the primer to find a low
copy target sequence among non-target sequences, a restriction
enzyme recognition site is preferably present in the deleted
sequence. Restriction digestion treatment prior to amplification
will then cleave non-target sequences. Preferably, the mutation
destroys a restriction enzyme recognition site such that the
wild-type sequence will be digested, but the mutant sequence no
longer contains the recognition site.
[0078] One of skill in the art would recognize that a restriction
enzyme fragmentation method can be modified by using a restriction
enzyme that cuts at a particular frequency or a particular site, or
by using multiple restriction enzymes. The choice of enzyme or
enzyme combinations is chosen to suit the target of interest in an
assay. Enzymes for fragmentation can be chosen by using a
restriction enzyme map of the region of interest. Such maps can be
readily generated by software programs well-known to those of skill
in the art.
[0079] Chemical fragmentation may include degradation by a nuclease
such as DNase or RNase which generate fragments having 3'-OH,
5'-OH, 3'-phosphate and 5'-phosphate ends; depurination or
depyrimidation with acid; the use of restriction enzymes;
intron-encoded endonucleases; DNA-based cleavage methods, such as
triplex and hybrid formation methods, that rely on the specific
hybridization of a nucleic acid segment to localize a cleavage
agent to a specific location in the nucleic acid molecule; or other
enzymes or compounds which cleave DNA at known or unknown locations
(see, for example, U.S. Pat. No. 6,495,320). It is possible to
depurinate or depyrimidinate the DNA, which is then fragmented in
the presence of a base (i.e., ".beta.-elimination") DNA can be
fragmented by oxidation, alkylation or free radical addition
mechanisms. Metal cations, which are often combined with organic
molecules which may function as chemical catalysts, for example
imidazole, are used for fragmenting RNA. This fragmentation is
preferably carried out in an alkaline medium and generates
fragments having 3'-phosphate ends. Chemical catalysts that may be
used for nucleic acid fragmentation include MOPS, HEPES, PIPES, and
bioorganic polyamines, such as spermine, spermidine and putrescine
(Bibille et al., 27 Nucleic Acids Res. 3931-3937 (1999)).
[0080] Different nucleic acid fragmentation techniques have been
described, for example, in Trawick et al., 98 Chem Rev. 939-960
(1998), Oivanen at al., 1998, 98 Chem Rev. 961-990 (1998) and
Laayoon, et al. U.S. Pat. No. 6,902,891. A method for fragmenting
and labeling RNA is described in WO88/04300A1, in which
fragmentation is carried out using RNA which possesses enzymatic
properties (ribozymes).
[0081] Physical fragmentation methods may involve subjecting the
DNA to a high shear rate. High shear rates may be produced, for
example, by moving DNA through a chamber or channel with pits or
spikes, or forcing the DNA sample through a restricted size flow
passage, e.g., an aperture having a cross sectional dimension in
the micron or submicron scale. Other physical methods include
sonication and nebulization. Combinations of physical and chemical
fragmentation methods may likewise be employed such as
fragmentation by heat and ion-mediated hydrolysis. See for example,
Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd Ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(2001).
[0082] Preferential cleavage can be achieved my other methods known
in the art such as the Maxam-Gilbert method. This method involves
degrading DNA at a specific base using chemical reagents. A. M.
Maxim et al., 65(1) Meth. in Enzym. 499-560 (1980). In general,
this method starts with end labeled DNA and cleaves by base
specific reagents. For example with guanine bases (the same
principle applies to all four bases), DNA of interest is
end-labeled (can be 5'- or 3'-end labeling). Then one kind of base
is modified, for example with dimethyl sulfate (DMS) to methylate
guanines. Conditions can be adjusted to achieve various frequencies
of methylation. Following methylation. a reagent such as piperidine
is added which causes loss of a methylated base and then breaks the
DNA backbone at the site of the lost base (the apurinic site).
Deletion Mutations
[0083] In one approach, a mutation specific primer is designed for
detecting a deletion mutation. Mutation specific primer can be
designed to span the deleted region such that the primer contains
wild-type sequence that lies 5' and 3' of the deleted region or the
complement thereof. Thus, the mutation specific primer cannot bind
to the wild-type sequence and cannot produce an amplicon.
[0084] Oligonucleotide primers may be designed for amplifying
regions of mutated nucleic acid. In one approach, a primer pair is
designed for detecting a deletion mutation. In one embodiment, the
primer pair is designed to hybridize to a specified segment of the
EGFR gene. The sequence of exemplary oligo primers are shown as
highlighted regions in FIGS. 2A and 2B (SEQ ID NOs:1 and 2).
Exemplary primer pairs for amplifying a region of the EGFR sequence
for the E746_A750del mutation use a forward primer (mutation
specific primer) with SEQ ID NO:1 (5'-CCCGTCGCTATCAAAACATC-3') and
a reverse primer with SEQ ID NO:2 (5'-ATGTGGAGATGAGCAGGGTCT-3'). In
this example, the mutation specific primer spans sequence that is
deleted in the mutated sequence. Thus, the primer cannot anneal to
the wild-type sequence due to the presence of the 15 base pairs.
Preferably, the primers with SEQ ID NOs:1 and 2 are each or both
used in conjunction with a restriction enzyme digestion treatment
with MseI which has a recognition site of TTAA. The mutation
specific primer in that example lies 5' and 3' of the 15 bp deleted
region, it cannot bind to the wild-type sequence, thus making the
primer mutation specific for the deletion mutation. FIGS. 1 and 2
illustrate this example of detecting the E746_A750del mutation in
the EGFR gene.
Insertion Mutations
[0085] In one approach, a mutation specific primer is designed for
detecting an insertion mutation. A mutation specific primer can be
designed to span all or a portion of the inserted region such that
the primer includes all or a part of the inserted region. A primer
could be designed to span the either or both junctions of the
inserted sequence, for example, the primer sequence would include a
portion of wild-type sequence that is adjacent to the inserted
sequence or the complement thereof. Thus, the mutation specific
primer is not complementary to the wild-type sequence and cannot
produce an amplicon.
[0086] Preferably, the insertion destroys a restriction enzyme
recognition site such that the wild-type sequence will be digested,
but the mutant sequence no longer contains the recognition site.
Restriction digestion treatment prior to amplification will then
cleave non-target sequences. Restriction digestion can also enhance
sensitivity by cleaving away sequence surrounding target nucleic
acid and facilitate amplification.
[0087] In one embodiment, a primer pair is designed to detect the
758 base pair insertion in exon 16 insertion in the APC gene. The
sequence of exemplary oligo primers are shown as highlighted
regions in FIG. 4 (SEQ ID NOs:3 and 4). Exemplary primer pairs for
amplifying a region of the APC sequence for an exon 16 (Miki, et
al., 52(3) Cancer Research 643-645 (1992)) insertion mutation use a
forward primer (mutation specific primer) with SEQ ID NO:3
(5'-CTTCCACAATGGTTGAACTAG-3') and a reverse primer (mutation
specific primer) with SEQ ID NO:4 (5'-CATCCATGTCCCTACAAAGG-3'). In
this example, both forward and reverse primers are mutation
specific because they lie within the insertion sequence.
Preferably, the primers with SEQ ID NOs:3 and 4 are each or both
used in conjunction with a restriction enzyme digestion treatment
with MnlI which has a recognition site of CCTC.
[0088] The mutation specific primer in that example lies within the
inserted region. Because the primers lie within the insertion
sequence, no amplification will occur unless the insertion is
present. In addition, there are MnlI restriction sites upstream and
downstream of the desired amplification product which will
facilitate amplification subsequent to digestion by removing
surrounding sequence. FIGS. 3 and 4 illustrate this example of
detecting the exon 16 insertion mutation in the APC gene.
[0089] In another embodiment, a primer pair is designed to detect
the 758 base pair insertion in exon 16 insertion in the APC gene.
The sequence of exemplary oligo primers are shown as highlighted
regions in FIG. 6 (SEQ ID NOs:5 and 6). Exemplary primer pairs for
amplifying a region of the APC sequence for an exon 16 insertion
mutation use a forward primer (mutation specific primer) with SEQ
ID NO:5 (5'-GAGCCATTTATACAGAAAGATG-3') and a reverse primer
(mutation specific primer) with SEQ ID NO:6
(5'-GAAATACCATTTGACCCAGC-3'). In this example, the forward primer
lies outside the insertion sequence and reverse primer lies inside
the insertion sequence. Both are mutation specific because an
amplicon will not be produced in the absence of the insertion
sequence. Preferably, the primers with SEQ ID NOs:5 and 6 are each
or both used in conjunction with a restriction enzyme digestion
treatment with MnlI which has a recognition site of CCTC.
[0090] One primer is upstream of the insertion site and the second
is within the insertion sequence. In addition, there are MnlI
restriction sites less than 20 bases upstream and downstream of the
desired amplification product which will facilitate amplification
of the target nucleic acid by removing surrounding sequence. The
wild-type APC sequence is heavily targeted by MnlI restriction
enzyme with a number of sites immediately downstream of the forward
primer. The insertion sequence in the APC exon 16 insertion mutant
contains a region devoid of MnlI restriction sites that is used as
the template for PCR. Digestion with MnlI prior to PCR eliminates
any linear amplification that may occur by forward primer binding
to the wild type APC gene. Fluorescent PCR can be performed using
one forward primer that binds to the APC gene just before the
insertion and one insertion specific reverse primer (unlabeled
forward, FAM labeled reverse) designed to specifically recognize
the insertion sequence in the region not containing MnlI
restriction sites. FIGS. 5 and 6 illustrate this example of
detecting the exon 16 insertion mutation in the APC gene.
Substitution Mutations
[0091] In one approach, a mutation specific primer is designed for
detecting one or more substitution mutations. A mutation specific
primer can be designed to include one or more substitutions. In a
preferred embodiment, the 3' nucleotide of the primer can be
designed such that it contains the mutated base pair and does not
bind, hybridize, or base pair, in the wild-type gene and thus
cannot elongate. In another preferred embodiment, the mutated base
pair is located at the -1 position at the 3'-end of a mutation
specific primer (i.e., the penultimate base).
[0092] Preferably, the one or more substitutions destroys a
restriction enzyme recognition site such that the wild-type
sequence will be digested, but the mutant sequence no longer
contains the recognition site. Restriction digestion treatment
prior to amplification will then cleave non-target sequences.
[0093] In further preferred embodiments, the mutated base pair in
the mutation specific primer is a locked nucleic acid (LNA). The
locked nucleic acid provides increased specificity by increasing
the melting temperature of the of a primer containing the
substitution base. This allows for the use of an increased
annealing temperature during amplification which decreases
amplification of wild type sequences.
[0094] In one embodiment, a primer pair is designed to detect the
L858R mutation in the EGFR gene. The sequence of exemplary oligo
primers are shown as highlighted regions in FIG. 8 (SEQ ID NOs:7
and 8). Exemplary primer pairs for amplifying a region of the EGFR
sequence for the L858R mutation use a forward primer (mutation
specific primer) with SEQ ID NO:7 (5'-TCACAGATTTTGGGCGG-3') and a
reverse primer with SEQ ID NO:8 (5'-CCTGGTGTCAGGAAAATGCT-3'). In
this example, the mutation specific primer contains the mutated
sequence at the terminal base. Thus, it will not properly anneal to
the wild-type sequence because the last base is not complementary.
Preferably, the primers with SEQ ID NOs:7 and 8 are each or both
used in conjunction with a restriction enzyme digestion treatment
with EaeI which has a recognition site of YGGCCR, where Y=C or T
and R=A or G. As shown in FIG. 8, the boxed MseI restriction sites,
TTAA, illustrate that a simultaneous reaction, such as a multiplex
PCR reaction, can be used to detect either or both the E746_A750del
and L858R mutations in the same reaction. Digestion with both MseI
and EaeI does not disrupt the L858R sequence of interest.
[0095] The mutation specific primer in that example includes the
mutated base pair sequence, a G, at its 3' end. Because the primer
is not complementary to the wild-type sequence, which contains a T,
elongation will not occur. The EaeI cut site allows cleavage of the
wild-type EGFR gene but is destroyed by the T-*G conversion. Thus,
when the L858R EGFR mutant is present, the recognition site is no
longer present and can no longer be digested by EaeI. FIGS. 7 and 8
illustrate this example of detecting the L858R mutation in the EGFR
gene.
Translocation Mutations
[0096] In one approach, a mutation specific primer is designed for
detecting one or more translocation mutations. A mutation specific
primer can be designed to span the junction of the translocation or
the complement thereof. A primer pair could be designed to so that
one primer is upstream of the translocation junction and the second
is downstream of the junction. Thus, when the primer pair is used
on wild-type sequence, no amplification products will be produced
because the locations of the primers relative to each other are
cannot be amplified. However, when the translocation is present,
the primers are in close enough proximity of each other such that
an amplification product can be produced. For example, the primer
can be designed to include a portion of the first gene and a
portion of the second gene, where the genes are located on
different chromosomes in wild-type form but are adjacent to one
another in the mutated form.
[0097] Preferably, one or more translocations destroys a
restriction enzyme recognition site such that the wild-type
sequence will be digested, but the mutant sequence no longer
contains the recognition site. Restriction digestion treatment
prior to amplification will then cleave non-target sequences.
[0098] In one embodiment, a primer pair is designed to detect the
TMPRSS2:ERG or translocation mutation of the TMPRSS2 and ERG genes.
The sequence of exemplary oligo primers are shown as higlighted
regions in FIG. 10 (SEQ ID NOs:9 and 10). Exemplary primer pairs
for amplifying a region of the TMPRSS2 and ERG sequences for the
TMPRSS2:ERG translocation mutation use a forward primer (mutation
specific primer) with SEQ ID NO:9 (5'-CGAGCTAAGCAGGAGGCGG-3') and a
reverse primer (mutation specific primer) with SEQ ID NO:10
(5'-GTCCATAGTCGCTGGAGGAG-3'). In this example, while both primers
anneal to wild-type sequences, they are mutation specific when used
in conjunction with each other because they will not produce an
amplification product unless the translocation is present in the
nucleic acid sample. Preferably, the primers with SEQ ID NOs:9 and
10 are each or both used in conjunction with a restriction enzyme
digestion treatment with FatI which has a recognition site of
CATG.
[0099] In another embodiment, a primer pair is designed to detect
the TMPRSS2:ETV1 translocation mutations of the TMPRSS2 and ETV1
genes. The sequence of exemplary oligo primers are shown as
highlighted regions in FIG. 12 (SEQ ID NOs:11 and 12). Exemplary
primer pairs for amplifying a region of the TMPRSS2 and ERG
sequences for the TMPRSS2:ETV1 translocation mutation use a forward
primer (mutation specific primer) with SEQ ID NO:11
(5'-CGAGCTAAGCAGGAGGCGG-3') and a reverse primer (mutation specific
primer) with SEQ ID NO:12 (5'-ACTTTCAGCCTGATAGTCTGG-3'). In this
example, while both primers anneal to wild-type sequences, they are
mutation specific when used in conjunction with each other because
they will not produce an amplification product unless the
translocation is present in the nucleic acid sample. Preferably,
the primers with SEQ ID NOs:11 and 12 are each or both used in
conjunction with a restriction enzyme digestion treatment with
HpyCH4VI which has a recognition site of TGCA.
[0100] In these embodiments, the Fat I and HpyCH4V cut sites allow
cleavage of the wild-type TMPRSS2, ERG, and ETV1 in the regions
that are absent in the fusion transcripts, essentially
"decontaminating" the sample of wild-type TMPRSS2 and ERG or ETV1
translocations. Because the forward and reverse primer sequences
are only both present in the fusion transcripts, only the nucleic
acids representing a fusion transcript will yield PCR products.
FIGS. 9 and 10 illustrate this example of detecting the TMPRSS2:ERG
translocation mutation.
Sample Preparation
[0101] The method may be performed using any sample containing
nucleic acid. Samples may be obtained by standard procedures and
may be used immediately or stored (e.g., the sample may be frozen
between about -15.degree. C. to about -100.degree. C.) for later
use. Samples may be obtained from patients suspected of having a
mutated nucleic acid sequence, for example from a tumor cell or
cancer cells. The presence of mutated nucleic acids in a sample can
be determined by amplifying cancer marker regions. Thus, any liquid
or solid material believed to contain cancer marker nucleic acids
can be an appropriate sample. Preferred sample tissues include
plasma, blood, bone marrow, body fluids, cerebrospinal fluid, urine
and others. Heparin is known to inhibit PCR (Beutler, et al.
BioTechniques 9:166, 1990), so samples containing heparin are not
ideal for the uses contemplated herein. Nucleic acid extraction
techniques that remove heparin are known in the art. These
techniques may be used to remove heparin from samples to make the
samples more suitable for amplification.
[0102] The sample may be processed to release or otherwise make
available a nucleic acid for detection as described herein. Such
processing may include steps of nucleic acid manipulation, e.g.,
preparing a cDNA by reverse transcription of RNA from the
biological sample. Thus, the nucleic acid to be amplified by the
methods of the invention may be genomic DNA, cDNA, single stranded
DNA or mRNA.
Oligonucleotides
[0103] Oligonucleotide primers may be approximately 15-100
nucleotides in length. Of the specific oligonucleotides provided
herein, additional variations of the primers comprise all or a
portion of the SEQ IDs described herein. Other preferred
oligonucleotide primers include an oligonucleotide sequence that
hybridizes to the complement of a 15-100 nucleotide sequence that
comprises the complement of all or a portion of the SEQ IDs
described herein. Such oligonucleotides may be substantially
purified.
Amplification of Nucleic Acids
[0104] Nucleic acid samples or isolated nucleic acids may be
amplified by various methods known to the skilled artisan.
Preferably, PCR is used to amplify mutated nucleic acids of
interest. In this method, two or more oligonucleotide primers that
flank or include, and anneal to opposite strands of a nucleic acid
of interest are repetitively annealed to their complementary
sequences, extended by a DNA polymerase (e.g., AmpliTaq Gold
polymerase), and heat denatured, resulting in exponential
amplification of the target nucleic acid sequences. Cycling
parameters can be varied, depending on the length of nucleic acids
to be extended. The skilled artisan is capable of designing and
preparing primers that are appropriate for amplifying a target
sequence in view of this disclosure. The length of the
amplification primers for use in the present invention depends on
several factors including the nucleotide sequence identity and the
temperature at which these nucleic acids are hybridized or used
during in vitro nucleic acid amplification. The considerations
necessary to determine a preferred length for an amplification
primer of a particular sequence identity are well known to the
person of ordinary skill. For example, the length of a short
nucleic acid or oligonucleotide can relate to its hybridization
specificity or selectivity.
[0105] Assay controls may be used in the assay for detecting a
mutated nucleic acid sequence. An internal positive amplification
control (IPC) can be included in the sample, utilizing
oligonucleotide primers and/or probes.
Detection of Amplified Nucleic Acids
[0106] Amplification of nucleic acids can be detected by any of a
number of methods well-known in the art such as gel
electrophoresis, column chromatography, hybridization with a probe,
or sequencing.
[0107] In one approach, sequences from two or more regions of
interest are amplified in the same reaction vessel. In this case,
the amplicon(s) could be detected by first size-separating the
amplicons then detection of the size-separated amplicons. The
separation of amplicons of different sizes can be accomplished by,
for example, gel electrophoresis, column chromatography, or
capillary electrophoresis. These and other separation methods are
well-known in the art. In one example, amplicons of about 10 to
about 150 base pairs whose sizes differ by 10 or more base pairs
can be separated, for example, on a 4% to 5% agarose gel, (a 2% to
3% agarose gel for about 150 to about 300 base pair amplicons) or a
6% to 10% polyacrylamide gel. The separated nucleic acids can then
be stained with a dye such as ethidium bromide and the size of the
resulting stained band or bands can be compared to a standard DNA
ladder.
[0108] In another embodiment, two or more regions of interest are
amplified in separate reaction vessels. If the amplification is
specific, that is, one primer pair amplifies for one region of
interest but not the other, detection of amplification is
sufficient to distinguish between the two types--size separation
would not be required.
[0109] In some embodiments, amplified nucleic acids are detected by
hybridization with a mutation-specific probe. Probe
oligonucleotides, complementary to a portion of the amplified
target sequence may be used to detect amplified fragments.
Amplified nucleic acids for each of the target sequences may be
detected simultaneously (i.e., in the same reaction vessel) or
individually (i.e., in separate reaction vessels). In preferred
embodiments, the amplified DNA is detected simultaneously, using
two distinguishably-labeled, gene-specific oligonucleotide probes,
one which hybridizes to the first target sequence and one which
hybridizes to the second target sequence.
[0110] The probe may be detectably labeled by methods known in the
art. Useful labels include, e.g., fluorescent dyes (e.g., Cy5.RTM.,
Cy3.RTM., FITC, rhodamine, lanthamide phosphors, Texas red), 32P,
35S, 3H, 14C, 125I, 131I, electron-dense reagents (e.g., gold),
enzymes, e.g., as commonly used in an ELISA (e.g., horseradish
peroxidase, beta-galactosidase, luciferase, alkaline phosphatase),
colorimetric labels (e.g., colloidal gold), magnetic labels (e.g.,
Dynabeads.TM.), biotin, dioxigenin, or haptens and proteins for
which antisera or monoclonal antibodies are available. Other labels
include ligands or oligonucleotides capable of forming a complex
with the corresponding receptor or oligonucleotide complement,
respectively. The label can be directly incorporated into the
nucleic acid to be detected, or it can be attached to a probe
(e.g., an oligonucleotide) or antibody that hybridizes or binds to
the nucleic acid to be detected.
[0111] A probe oligonucleotide, complementary to the amplified
region of nucleic acid, is used to detect the amplification of
mutated nucleic acids. The probe may be detectably labeled by
methods known in the art. The binding of a probe to the amplified
region of the mutated nucleic acid may be determined by
hybridization as is well known in the art. Hybridization may be
detected in real time or in non-real time.
[0112] One general method for real time PCR uses fluorescent probes
such as the TaqMan.RTM. probes, molecular beacons and scorpions.
Real-time reverse-transcriptase (RT) PCR quantitates the initial
amount of the template with more specificity, sensitivity and
reproducibility, than other forms of quantitative reverse
transcriptase PCR, which detect the amount of final amplified
product. Real-time RT-PCR does not detect the size of the amplicon.
The probes employed in TaqMan.RTM. and molecular beacon
technologies are based on the principle of fluorescence quenching
and involve a donor fluorophore and a quenching moiety.
[0113] In a preferred embodiment, the detectable label is a
fluorophore. The term "fluorophore" as used herein refers to a
molecule that absorbs light at a particular wavelength (excitation
frequency) and subsequently emits light of a longer wavelength
(emission frequency). The term "donor fluorophore" as used herein
means a fluorophore that, when in close proximity to a quencher
moiety, donates or transfers emission energy to the quencher. As a
result of donating energy to the quencher moiety, the donor
fluorophore will itself emit less light at a particular emission
frequency that it would have in the absence of a closely positioned
quencher moiety.
[0114] The term "quencher moiety" as used herein means a molecule
that, in close proximity to a donor fluorophore, takes up emission
energy generated by the donor and either dissipates the energy as
heat or emits light of a longer wavelength than the emission
wavelength of the donor. In the latter case, the quencher is
considered to be an acceptor fluorophore. The quenching moiety can
act via proximal (i.e., collisional) quenching or by Forster or
fluorescence resonance energy transfer ("FRET"). Quenching by FRET
is generally used in TaqMan.RTM. probes while proximal quenching is
used in molecular beacon and scorpion type probes.
[0115] In proximal quenching (a.k.a. "contact" or "collisional"
quenching), the donor is in close proximity to the quencher moiety
such that energy of the donor is transferred to the quencher, which
dissipates the energy as heat as opposed to a fluorescence
emission. In FRET quenching, the donor fluorophore transfers its
energy to a quencher which releases the energy as fluorescence at a
higher wavelength. Proximal quenching requires very close
positioning of the donor and quencher moiety, while FRET quenching,
also distance related, occurs over a greater distance (generally
1-10 nm, the energy transfer depending on R-6, where R is the
distance between the donor and the acceptor). Thus, when FRET
quenching is involved, the quenching moiety is an acceptor
fluorophore that has an excitation frequency spectrum that overlaps
with the donor emission frequency spectrum. When quenching by FRET
is employed, the assay may detect an increase in donor fluorophore
fluorescence resulting from increased distance between the donor
and the quencher (acceptor fluorophore) or a decrease in acceptor
fluorophore emission resulting from increased distance between the
donor and the quencher (acceptor fluorophore).
[0116] Suitable fluorescent moieties include the following
fluorophores known in the art: [0117]
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid [0118]
acridine and derivatives: [0119] acridine [0120] acridine
isothiocyanate [0121] Alexa Fluor.RTM. 350, Alexa Fluor.RTM. 488,
Alexa Fluor.RTM. 546, Alexa Fluor.RTM. 555, Alexa Fluor.RTM. 568,
Alexa Fluor.RTM. 594, Alexa Fluor.RTM. 647 (Molecular Probes)
[0122] 5-(2'-aminoethy)aminonaphthalene-1-sulfonic acid (EDANS)
[0123] 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5
disulfonate (Lucifer Yellow VS) [0124]
N-(4-anilino-1-naphthyl)maleimide [0125] anthranilamide [0126]
Black Hole Quencher.TM. (BHQ.TM.) dyes (biosearch Technologies)
[0127] BODIPY.RTM. R-6G, BOPIPY.RTM. 530/550, BODIPY.RTM. FL [0128]
Brilliant Yellow [0129] coumarin and derivatives: [0130] coumarin
[0131] 7-amino-4-methylcoumarin (AMC, Coumarin 120) [0132]
7-amino-4-trifluoromethylcouluarin (Coumarin 151) [0133] Cy2.RTM.,
Cy3.RTM., Cy3.5.RTM., Cy5.RTM., Cy5.5.RTM. [0134] cyanosine [0135]
4',6-diaminidino-2-phenylindole (DAPI) [0136] 5',
5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red) [0137]
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin [0138]
diethylenetriamine pentaacetate [0139]
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid [0140]
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [0141]
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride) [0142] 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL)
[0143] 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC)
[0144] Eclipse.TM. (Epoch Biosciences Inc.) [0145] eosin and
derivatives: [0146] eosin [0147] eosin isothiocyanate [0148]
erythrosin and derivatives: [0149] erythrosin B [0150] erythrosin
isothiocyanate [0151] ethidium [0152] fluorescein and derivatives:
[0153] 5-carboxyfluorescein (FAM) [0154]
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) [0155]
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) [0156]
fluorescein [0157] fluorescein isothiocyanate (FITC) [0158]
hexachloro-6-carboxyfluorescein (HEX) [0159] QFITC (XRITC) [0160]
tetrachlorofluorescein (TET) [0161] fluorescamine [0162] IR144
[0163] IR1446 [0164] Malachite Green isothiocyanate [0165]
4-methylumbelliferone [0166] ortho cresolphthalein [0167]
nitrotyrosine [0168] pararosaniline [0169] Phenol Red [0170]
B-phycoerythrin, R-phycoerythrin [0171] o-phthaldialdehyde [0172]
Oregon Green.RTM. [0173] propidium iodide [0174] pyrene and
derivatives: [0175] pyrene [0176] pyrene butyrate [0177]
succinimidyl 1-pyrene butyrate [0178] QSY.RTM. 7, QSY.RTM. 9,
QSY.RTM. 21, QSY.RTM. 35 (Molecular Probes) [0179] Reactive Red 4
(Cibacron.RTM. Brilliant Red 3B-A) [0180] rhodamine and
derivatives: [0181] 6-carboxy-X-rhodamine (ROX) [0182]
6-carboxyrhodamine (R6G) [0183] lissamine rhodamine B sulfonyl
chloride [0184] rhodamine (Rhod) [0185] rhodamine B [0186]
rhodamine 123 [0187] rhodamine green [0188] rhodamine X
isothiocyanate [0189] sulforhodamine B [0190] sulforhodamine 101
[0191] sulfonyl chloride derivative of sulforhodamine 101 (Texas
Red) [0192] N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA) [0193]
tetramethyl rhodamine [0194] tetramethyl rhodamine isothiocyanate
(TRITC) [0195] riboflavin [0196] rosolic acid [0197] terbium
chelate derivatives
[0198] Other fluorescent nucleotide analogs can be used, see, e.g.,
Jameson, 278 Meth. Enzymol. 363-390 (1997); Zhu, 22 Nucl. Acids
Res. 3418-3422 (1994). U.S. Pat. Nos. 5,652,099 and 6,268,132 also
describe nucleoside analogs for incorporation into nucleic acids,
e.g., DNA and/or RNA, or oligonucleotides, via either enzymatic or
chemical synthesis to produce fluorescent oligonucleotides. U.S.
Pat. No. 5,135,717 describes phthalocyanine and
tetrabenztriazaporphyrin reagents for use as fluorescent
labels.
[0199] The detectable label can be incorporated into, associated
with or conjugated to a nucleic acid. Label can be attached by
spacer arms of various lengths to reduce potential steric hindrance
or impact on other useful or desired properties. See, e.g.,
Mansfield, 9 Mol. Cell. Probes 145-156 (1995).
[0200] Detectable labels can be incorporated into nucleic acids by
covalent or non-covalent means, e.g., by transcription, such as by
random-primer labeling using Klenow polymerase, or nick
translation, or amplification, or equivalent as is known in the
art. For example, a nucleotide base is conjugated to a detectable
moiety, such as a fluorescent dye, e.g., Cy3.RTM. or Cy50 and then
incorporated into genomic nucleic acids during nucleic acid
synthesis or amplification. Nucleic acids can thereby be labeled
when synthesized using Cy3.RTM.- or Cy5.RTM.-dCTP conjugates mixed
with unlabeled dCTP.
[0201] Nucleic acid probes can be labeled by using PCR or nick
translation in the presence of labeled precursor nucleotides, for
example, modified nucleotides synthesized by coupling
allylamine-dUTP to the succinimidyl-ester derivatives of the
fluorescent dyes or haptens (such as biotin or digoxigenin) can be
used; this method allows custom preparation of most common
fluorescent nucleotides, see, e.g., Henegariu, 18 Nat. Biotechnol.
345-348 (2000).
[0202] Nucleic acid probes may be labeled by non-covalent means
known in the art. For example, Kreatech Biotechnology's Universal
Linkage System.RTM. (ULS.RTM.) provides a non-enzymatic labeling
technology, wherein a platinum group forms a co-ordinative bond
with DNA, RNA or nucleotides by binding to the N7 position of
guanosine. This technology may also be used to label proteins by
binding to nitrogen and sulphur containing side chains of amino
acids. See, e.g., U.S. Pat. Nos. 5,580,990; 5,714,327; and
5,985,566; and European Patent No. 0539466.
[0203] The binding of a probe to the marker sequence flanking the
tandem repeat region may be determined by hybridization as is well
known in the art. Hybridization may be detected in real time or in
non-real time.
[0204] TaqMan.RTM. probes (Heid, et al., 1996) use the fluorogenic
5' exonuclease activity of Taq polymerase to measure the amount of
target sequences in cDNA samples. TaqMan.RTM. probes are
oligonucleotides that contain a donor fluorophore usually at or
near the 5' base, and a quenching moiety typically at or near the
3' base. The quencher moiety may be a dye such as TAMRA or may be a
non-fluorescent molecule such as 4-(4-dimethylaminophenylazo)
benzoic acid (DABCYL). See Tyagi, et al., 16 Nature Biotechnology
49-53 (1998). When irradiated, the excited fluorescent donor
transfers energy to the nearby quenching moiety by FRET rather than
fluorescing. Thus, the close proximity of the donor and quencher
prevents emission of donor fluorescence while the probe is
intact.
[0205] TaqMan.RTM. probes are designed to anneal to an internal
region of a PCR product. When the polymerase (e.g., reverse
transcriptase) replicates a template on which a TaqMan.RTM. probe
is bound, its 5' exonuclease activity cleaves the probe. This ends
the activity of quencher (no FRET) and the donor fluorophore starts
to emit fluorescence which increases in each cycle proportional to
the rate of probe cleavage. Accumulation of PCR product is detected
by monitoring the increase in fluorescence of the reporter dye
(note that primers are not labeled). If the quencher is an acceptor
fluorophore, then accumulation of PCR product can be detected by
monitoring the decrease in fluorescence of the acceptor
fluorophore.
[0206] TaqMan.RTM. assay uses universal thermal cycling parameters
and PCR reaction conditions. Because the cleavage occurs only if
the probe hybridizes to the target, the fluorescence detected
originates from specific amplification. The process of
hybridization and cleavage does not interfere with the exponential
accumulation of the product. One specific requirement for
fluorogenic probes is that there be no G at the 5' end. A `G`
adjacent to the reporter dye quenches reporter fluorescence even
after cleavage.
[0207] Other methods of probe hybridization detected in real time
can be used for detecting amplification of mutated nucleic acids.
For example, the commercially available MGB Eclipse.TM. probes
(Epoch Biosciences), which do not rely on a probe degradation can
be used. MGB Eclipse.TM. probes work by a hybridization-triggered
fluorescence mechanism. MGB Eclipse.TM. probes have the Eclipse.TM.
Dark Quencher and the MGB positioned at the 5'-end of the probe.
The fluorophore is located on the 3'-end of the probe. When the
probe is in solution and not hybridized, the three dimensional
conformation brings the quencher into close proximity of the
fluorophore, and the fluorescence is quenched. However, when the
probe anneals to a target sequence, the probe is unfolded, the
quencher is moved from the fluorophore, and the resultant
fluorescence can be detected.
[0208] Suitable donor fluorophores include 6-carboxyfluorescein
(FAM), tetrachloro-6-carboxyfluorescein (TET),
2'-chloro-7'-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC) and the
like. Suitable quenchers include tetra-methylcarboxyrhodamine
(TAMRA) 4-(4-dimethylaminophenylazo) benzoic acid ("DABCYL" or a
DABCYL analog) and the like. Tetramethylrhodamine (TMR) or
5-carboxyrhodamine 6G (RHD) may be combined as donor fluorophores
with DABCYL as quencher. Multiplex TaqMan.RTM. assays can be
performed using multiple detectable labels each comprising a
different donor and quencher combination. Probes for detecting
amplified sequence in real time may be stored frozen (-10.degree.
to -30.degree. C.) as 100 M stocks. TaqMan.RTM. probes are
available from Applied BioSystems (4316032).
[0209] In a preferred embodiment, real time PCR is performed using
TaqMan.RTM. probes in combination with a suitable
amplification/analyzer such as the ABI Prism 7900HT Sequence
Detection System. The ABI PRISM.RTM. 7900HT Sequence Detection
System is a high-throughput real-time PCR system that detects and
quantitates nucleic acid sequences. Briefly, TaqMan.RTM. probes
specific for each allele are included in the PCR assay. These
probes contain a reporter dye at the 5' end and a quencher dye at
the 3' end. Each allele specific probe is conjugated with a
different fluorescent reporter dye. During PCR, the fluorescently
labeled probes bind specifically to their respective target
sequences; the 5' nuclease activity of Taq polymerase cleaves the
reporter dye from the probe and a fluorescent signal is generated.
The increase in fluorescence signal is detected only if the target
sequence is complementary to the probe and is amplified during PCR.
A mismatch between probe and target greatly reduces the efficiency
of probe hybridization and cleavage. The ABI Prism 7700HT or 7900HT
Sequence detection System measures the increase in fluorescence
during PCR thermal cycling, providing "real time" detection of PCR
product accumulation.
[0210] Real Time detection on the ABI Prism 7900HT or 7900HT
Sequence Detector monitors fluorescence and calculates the measure
of reporter signal, or Rn value, during each PCR cycle. The
threshold cycle, or Ct value, is the cycle at which fluorescence
intersects the threshold value. The threshold value is determined
by the sequence detection system software or manually.
[0211] To minimize the potential for cross contamination, reagent
and mastermix preparation, specimen processing and PCR setup, and
amplification and detection are all carried out in physically
separated areas. In addition, Uracil-N-Glycosylase is utilized
(along with the incorporation of Uracil into PCR amplicons) to
eliminate carry over contamination.
[0212] The following examples serve to illustrate the present
invention. These examples are in no way intended to limit the scope
of the invention.
Example 1
E746_A750 Deletion
[0213] Background:
[0214] Somatic mutations in the tyrosine kinase (TK) domain of the
EGFR gene are associated with clinical response to TK inhibitors in
patients with non-small cell lung cancer (NSCLC). An assay that
detects such mutations in plasma provides a noninvasive procedure
to assess suitability for TK inhibitor therapy. Described below are
1) the development of a sensitivity assay to detect the E746_A750
deletion (E746_A750del) in the TK domain of EGFR in plasma; and 2)
optimization of the assay for plasma DNA extraction.
[0215] Methods:
[0216] The assay uses MseI to specifically digest wild-type (WT)
genomic DNA (gDNA) to reduce non-specific amplification. After
digestion, samples are PCR-amplified using one unlabeled primer and
one FAM-labeled primer spanning the E746_A750 deletion region. The
fluorescence signal is detected with an automated genetic analyzer.
Serial dilution studies were conducted using H1650 gDNA, which is a
E746_A750 deletion cell line) diluted in WT gDNA after MseI
digestion. To assess detection of the deletion in plasma, 3-4 mL of
whole blood was spiked with 1-37 ng H1650 gDNA; gDNA from the
separated plasma was then extracted by silica column/2-propanol
precipitation, digested with MseI, and amplified as above. Several
extraction methods (silica column, magnetic bead,
phenol:chloroform, and 2-propanol precipitation) were evaluated
using pooled plasma samples and phosphate buffer solution (PBS)
spiked with 10-350 ng of gDNA.
[0217] Results:
[0218] Using a combined approach of digesting the WT EGFR allele
followed by deletion-specific fluorescent PCR, the equivalent of
circa 1 copy of the E746_A750 deletion (10 pg gDNA) diluted to
0.001% could be detected. Furthermore, the E746_A750 deletion was
successfully detected in 1/5 the final DNA volume (5 .mu.l) in all
spiked blood samples. In the DNA extraction method evaluation, the
magnetic bead-based method yielded the highest percent recovery of
gDNA from PBS (69% recovery of the 10 ng sample). Phenol:chloroform
extraction gave the highest yield with pooled plasma samples.
[0219] Conclusions:
[0220] The combination of an optimized DNA extraction method,
clearing the plasma DNA sample of amplifiable WT DNA by restriction
digestion, and mutation-specific fluorescent PCR provides a highly
sensitive assay for detection of somatic mutations in plasma.
Example 2
Preparation of and Sensitivity Studies for Detection of EGFR
Mutations
[0221] Serial dilution studies were conducted using H1650 gDNA
(E746_A750 deletion mutation cell line) diluted in wild-type gDNA
with no treatment of after MseI digestion. For each dilution, 10 pg
of H1650 gDNA. H1650 is a heterozygous cell line, thus, 50% of the
DNA contributes to the deletion allele and 50% to the wild-type
allele) was spiked into wild-type gDNA at varying concentrations to
yield 0.001%-10% of the exon 19 deletion in the background of
wild-type allele. Nucleic acid is spiked into a sample in order to
control the amount of target nucleic acid in a sample and test the
sensitivity of the assay to detect various amounts, in particular
low amounts, of target nucleic acid. The results show that peaks
were detected corresponding to the deletion mutant at levels of
0.01% in non-treated samples and at least 0.001% when digested with
MseI. These results are presented in Table 1.
TABLE-US-00001 TABLE 1 Exon 19 deletion tumor DNA spiked into
purified DNA wild-type DNA % Exon 19 DNA Amount per Peak Detected
deletion reaction Concentration No MseI allele Wild-Type Exon 19
Deletion Treatment Treated 10.00% 45 pg 10 pg (50% E746_A750del)
Yes Yes 1.000% 495 pg 10 pg (50% E746_A750del) Yes No 0.100% 5 ng
10 pg (50% E746_A750del) Yes Yes 0.010% 50 ng 10 pg (50%
E746_A750del) Yes Yes 0.001% 500 ng 10 pg (50% E746_A750del) No Yes
0.000% 500 ng 0 pg No No *10 pg of tumor DNA was spiked into
wild-type DNA at varying concentrations, but since 1 allele is
wild-type, only half of the tumor DNA contributes to exon 19
deletion DNA and the other half to the wild-type allele
[0222] To assess detection of the deletion in plasma, 3-4 mL whole
blood was spiked with 1-37 ng H1650 gDNA; gDNA from the separated
plasma was then extracted (QiaAmp DNA Blood Midi Kit), further
concentrated by ethanol or isopropanol precipitation, and digested
with MseI. The digested DNA was then subjected to fluorescent PCR
using deletion-specific primers.
TABLE-US-00002 TABLE 2 Exon 19 deletion tumor DNA spiked into blood
Peak Detected Amt of Spiked No Precipitation Method H1650 gDNA
Treatment Mse I Treated Ethanol Precipitation 37 ng No Yes 1 ng No
No Isopropanol Precipitation 37 ng NT Yes 1 ng NT Yes *All samples
were first extracted using the QiaAmp DNA Blood Midi kit, then
further precipitated by ethanol or isopropanol precipitation NT,
Not tested
Example 3
Method Comparison of EGFR Mutation Detection
[0223] A method comparison was performed to demonstrate the
sensitivity of the EGFR mutation detection assay disclosed herein
(also referred to as the Sanders method) which is designed to be
able to detect mutations in plasma from NSCLC patients. In this
study, the method disclosed herein was compared with two other
methods (Asano, 2006 and Ohnishi, 2006) that claim high sensitivity
for detecting E746_A750del. The Asano and Ohnishi methods were
performed as described in the respective publications but included
a fluorescent label on the forward primer. Amplification products
were then analyzed by capillary electrophoresis for fluorescent
detection of the PCR fragments using an ABI 3100 Genetic Analyzer.
Table 3 shows the expected and observed fragment sizes for each
fragment.
TABLE-US-00003 TABLE 3 Expected and observed fragment sizes Method
Fragment Expected Size Observed Size Sanders Wild-Type EGFR 197 194
E746_A750del 153 151 Asano Wild-Type EGFR 138 135 E746_A750del 123
120 Ohnishi E746_A750del 133 138 *Due to the mobility shift of PCR
primer/products fluorescently labeled with FAM, some of the
amplicons are slightly shifted from the expected size in the ABI
3100 Genetic Analyzer.
[0224] The assay disclosed herein has been previously detected as
little as 10 pg of H1650 cell line DNA (circa 1 copy of the
E746_A750del mutation) at as low as 0.001% in the background of the
wild-type EGFR gene. Therefore, all three methods (Sanders, Asano
and Ohnishi) were tested for their ability to detect 10 pg of H1650
cell line DNA at levels ranging from 0.0005% to 10% in the
background of the wild-type EGFR gene (Table 4).
TABLE-US-00004 TABLE 4 Sensitivity of Sanders, Asano, and Ohnishi
methods for detecting the E746_A750del mutation. Sanders Method
Asano Method Ohnishi Method Wild-Type Deletion Peak Wild-Type
Deletion Peak Deletion Peak Peak Intensity Intensity Peak Intensity
Intensity % H1650 DNA Intensity (1/5) (120 bp)* (135 bp)* (138 bp)*
10% 937 0 5811 0 0 1% 989 166 7209 195 0 0.10% 1395 152 2685 1746 0
0.01% 520 1395 1004 3682 0 0.001% 820 796 97 7087 0 0.0005% 1184
2321 0 7338 167 0.0003% 699 3942 NT NT NT 0.0002% 803 3324 NT NT NT
0.0001% 0 3798 0 4603 NT 0% (5 .mu.g) 0 2287 0 3836 NT 0% (50 ng) 0
0 0 5280 0 *Sizes indicated are the actual size NT = Not Tested
[0225] The results of this comparison indicate that the Sanders
method detects circa 1 copy of the E746_A750del mutation in as
little as 0.0005% in the background of the wild-type EGFR gene.
(6.6 picograms (pg) is equivalent to one copy, so 10 pg is 1.5
copies, thus, circa is used to indicate the approximate copy
number.) The Asano method demonstrated strong peaks at 0.01-10%
levels and a weak peak at 0.001%, but no detectable amplification
at 0.0005%. The Ohnishi method was unable to detect circa 1 copy of
the E746_A750del mutation as determined by the absence of any peak
from 0.001%-10% levels. However, a weak peak was observed in the
0.0005% sample, although this is most likely attributed to
background amplification of the wild-type EGFR gene due to the very
high levels of wild-type DNA present in the sample.
[0226] The Asano and Ohnishi methods were also tested for their
ability to detect the E746_A750del mutation at high and low copy
numbers without interfering wild-type DNA spike into the sample.
Table 5 shows that the Asano method was successful at detecting
both a high copy numbers (850 pg=130 copies) and low copy numbers
(10 pg=.about.1 copy) of the mutation, as also demonstrated above.
However, while the Ohnishi method successfully detected high copy
numbers of the mutation, it failed at detecting low copy
numbers.
TABLE-US-00005 TABLE 5 Detection of low and high copy numbers of
the E746_A750del mutation. Asano Method Ohnishi Method Deletion
Peak Wild Type Deletion Peak H1650 DNA Intensity Peak Intensity
Intensity (pg/rxn) (120 bp)* (135 bp)* (138 bp)* 850 7345 0 2008 10
2422 0 0 *Sizes indicated are the actual size
[0227] The results presented in this study confirm that the Sanders
assay for detection of the exon 19 EGFR deletion (E746_A750del)
utilizing restriction digestion followed by deletion specific
fluorescent PCR, demonstrates superior sensitivity over methods in
the prior art, in particular, the methods described by Asano and
Ohnishi.
Example 4
[0228] Detection of E746_A750del Mutation in Plasma of a NSCLC
Patient
[0229] The assay tested two separate DNA extractions from plasma of
a single NSCLC patient. Each extraction was digested with MseI and
subsequently split into 11 separate PCR reactions. Two reactions
yielded positive results as shown in Table 6 below.
TABLE-US-00006 TABLE 6 E746_A750del Mutation in the Plasma of a
NSCLC Patient Peak Intensity 500 .mu.l Plasma 350 .mu.l Plasma
Primer set Aliquot# 1:10 Undiluted 1:10 Undiluted E746_A750del 1 0
0 53 163 2 0 0 0 0 3 3568 7952 0 0 4 0 0 0 0 5 0 0 0 0 6 0 0 0 0 7
0 0 0 0 8 0 0 0 0 9 3971 7359 0 0 10 0 0 0 0 11 0 0 2518 6469 WT 1
0 -- 0 --
Example 5
DNA Extraction Comparison
[0230] Detection of rare mutations in plasma is a difficult feat
and is dependant on both the ability to detect very low amounts of
the mutation among large amounts of normal DNA and the ability to
successfully recover the mutation from the patient plasma sample.
Using a high quality DNA extraction method in conjunction with the
methods and compositions provided herein, particularly combining
non-target fragmentation with mutation specific primers further
increases the ability to detect low copy target nucleic acid in
patient samples, particularly plasma.
[0231] To determine an optimal method of DNA extraction, eight DNA
extraction methods were evaluated to find a method that is superior
for obtaining high yield DNA as well as providing DNA that is
amenable to the detection methods provided herein. Six plasma
samples for each extraction method were spiked with various nucleic
acids including every plasma sample being spiked with the EGFR exon
19 deletion (H1650 cell line gDNA) for subsequent evaluation of
detection. All six plasma samples were extracted using the eight
methods that included two lysis methods using magnetic bead based
methods (Agencourt Genfind.TM. and Ambion MagMax.TM.), two column
based methods (Qiagen QIAmp DNA Blood Mini Kit and the automated
Corbett DNA Xtractor.TM.), two isopropanol precipitation based
methods (Gentra Puregene.TM. and Roche Cobas.RTM.), and two
phenol:chloroform based methods (standard method and with Eppendorf
Phase Lock Gels.RTM.). The resulting yield was determined by
picogreen fluorescent assay (Invitrogen Quant-iT.TM. PicoGreen.RTM.
dsDNA Quantitation Assay) and the mean percent recovery of the six
plasma samples was calculated for each method. Comparison of these
eight DNA extraction methods revealed that three methods were
clearly more efficient at DNA recovery than the other five (FIG.
14). These methods included Agencourt Genfind.TM., Roche Cobas.RTM.
and phenol:chloroform extraction using Eppendorf Phase Lock
Gels.RTM..
[0232] Ambion MagMax.TM. is an isolation kit which can be used to
isolate RNA or DNA from serum, plasma, or any other biofluid.
Agencourt Genfind.TM. is a DNA isolation for blood, serum, or
plasma samples. Both involve sample disruption with a lysis reagent
followed by binding of the nucleic acid to magnetic beads
(proprietary chemistry). The beads are then washed with a series of
buffers to reduce and/or eliminate proteins and other contaminants
from the sample in order to purify the nucleic acid. The nucleic
acid is then eluted off the beads to yield the final DNA
sample.
[0233] QIAamp DNA Blood Mini Kit and Corbett DNA Xtractor.TM. are
both column based methods. The QIAamp procedure used in these
studies was manual while the Corbett was an automated system. Both
involve disruption of the sample with a lysis reagent followed by
binding to a silica column. The column containing bound nucleic
acid is washed with a series of wash buffers and the nucleic acid
is then eluted in the last step with elution buffer to yield the
final sample.
[0234] Gentra Puregene.TM. and Roche Cobas.RTM. methods are crude
extractions and both incorporate treatment of plasma with a lysis
reagent to disrupt the sample followed by isopropanol precipitation
of DNA.
[0235] Phenol:Chloroform extraction involves separation of organic
and aqueous phases of the plasma. The aqueous phase containing
nucleic acid is isolated and re-extracted once more with
phenol:chloroform. The aqueous phase is again isolated and the DNA
is purified from this phase by isopropanol precipitation.
[0236] Phenol:Chloroform extraction using Phase Lock Gels.RTM.
employs the phenol:chloroform procedure as described above, but
once separated the aqueous and organic phases are separated by a
solid gel. This allows increased recovery of the aqueous phase
without contamination from the organic phase which can lead to
inhibition of PCR.
[0237] Further evaluations were performed to identify the best
method from the three yielding the highest amounts of DNA. For this
comparison, 18% of the final DNA sample from each extraction was
subjected to our EGFR mutation detection assay as described in the
Examples above. The mean peak intensity of the PCR products
obtained corresponding to the E746_A750del EGFR mutation were
calculated as an indicator of DNA quality and recovery of the
spiked mutation (FIG. 15). Peak intensity values indicated that of
the three methods tested, the Agencourt Genfind.TM. method provided
the most robust amplification of spiked mutation.
[0238] Once the superior method was identified, further evaluation
of the six nucleic acid carrier conditions was performed to
determine if either condition facilitated recovery of the spiked
mutation. These nucleic acid carrier conditions included use of RNA
carrier or no carrier at low and high (100 nanograms (ng) spiked
normal DNA) plasma DNA levels and the use of normal DNA as a
carrier (395 ng and 778 ng). To determine optimal nucleic acid
carrier conditions, dilutions of each sample were analyzed using
our detection method. The concentration of mutant DNA in the
extracted samples was first estimated based on the calculated
percent recovery for each sample. The equivalent of 15 pg of H1650
DNA was used as the starting point and was further diluted 1:2 and
1:4. All three dilutions were analyzed for the presence of
detectable spiked EGFR mutation. The samples employing RNA carrier
were the only ones that detected the mutation in the 1:4 dilution
indicating that spiking RNA carrier during the extraction
facilitates recovery of the mutation present in the plasma sample
(FIG. 16). FIG. 16 provides the results from evaluation of nucleic
acid spiking conditions. The six nucleic acid carrier conditions
include 1) no carrier, 2) no carrier plus 100 ng of normal DNA, 3)
1 .mu.g of RNA carrier, 4) 1 .mu.g of RNA carrier plus 100 ng of
normal DNA, 5) 395 ng of normal DNA as a carrier, and 6) 778 ng of
normal DNA as a carrier. Plasma samples spiked with 100 ng of
normal DNA were included to represent patient plasma samples
containing high amounts of DNA and were spiked immediately after
thawing of the plasma sample. The two samples containing 395 ng and
778 ng of normal DNA were spiked following sample lysis during the
extraction, the point at which nucleic acid carrier is to be added.
Columns represent the mean peak intensity of E746_A750del PCR
product.
[0239] The experiments performed in this study identified an
optimal method for obtaining high yield DNA from plasma samples
that is amenable to detection of rare mutations. This method was
further improved by identifying nucleic acid spiking conditions
that facilitate the recovery of mutations from the sample. Thus to
improve the ability to recover rare mutations in the plasma and to
successfully detect such mutations, it was determined that the
Agencourt Genfind.TM. method with a modification to include
addition of RNA carrier to the sample lysate is the choice
method.
[0240] While Agencourt Genfind.TM. was the optimal extraction
method in these experiments, any of the extraction methods tested
can be used and are provide as exemplary extraction methods that
can be used in conjunction with the methods and compositions
provided herein. In different hands, results may vary, but all are
acceptable methods.
Example 6
Detection Sensitivity of L858R in Wild-Type Background
[0241] DNA samples were initially digested with restriction enzymes
(New England Biolabs) to cleave TTAA (MseI) and TGGCCA (MscI)
recognition sites targeting wild type EGFR sequences (Mse I for
singleplex E746_A750del; Msc I for singleplex L858R; Mse I and Msc
I for multiplex reactions) for 2 hours at 37.degree. C. followed by
20 minute inactivation at 65.degree. C. Following restriction
digestion, samples were amplified with AccuPrime.TM. Taq DNA
Polymerase (Invitrogen) using E746_A750del and/or L858R
mutation-specific primers (E746_A750del forward: 5'-[6FAM] CCC GTC
GCT ATC AAA ACA TC-3' (SEQ ID NO:1); E746_A750del reverse: 5'-ATG
TGG AGA TGA GCA GGG TCT-3' (SEQ ID NO:2); L858R forward: [6FAM] TCA
CAG ATT TTG GGC GG-3' (SEQ ID NO:7); L858R reverse: CCT GGT GTC AGG
AAA ATG CT-3' (SEQ ID NO:8)). In each set, the forward primer was
labeled with 5'-6FAM. For added specificity, the L858R forward
primer contained a locked nucleic acid (LNA) in the -1 position
corresponding to the mutated base. Thermocycling conditions were as
follows: denatured at 95.degree. C. for 5 min; amplified with 40
cycles of 94.degree. C. for 40 seconds, 55.degree. C. (E746_A750del
singleplex PCR) or 61.7.degree. C. (L858R singleplex PCR and
multiplex PCR) for 1 minute, 72.degree. C. for 1 minute; final
extension at 72.degree. C. for 7 minutes. E746_A750del PCR product
yielded an expected size of 153 bp and L858R PCR product yielded an
expected size of 113 bp.
TABLE-US-00007 TABLE 7 Detection of L858R Mutation in the
Background of Wild type EGFR. Mean Peak Intensity.sup.1 % L858R
(110 bp).sup.2 0.001% 463 0.0005% 412 0.0003% -- 0.0002% -- 0% (2.5
ug/rxn) -- .sup.1Peak intensities <200 RFU are not reported
.sup.2Size represents observed L858R peak size on ABI 3100 Genetic
Analyzer
[0242] Several other mutation specific primers for detecting the
L858R mutation were tested but were not effective in determining
the presence of the mutant nucleic acid. The primers are provided
in the table below:
TABLE-US-00008 TABLE 8 L858R Mutation Specific Primers. Mutant Base
as a Locked SEQ Nucleic Acid ID NO: Primer Sequence (Yes or No) 13
ATCACAGATTTTGGGCG Yes 14 CAAGATCACAGATTTTGGGCG No 15
ATGTCAAGATCACAGATTTTGGGCG No 16 AGATCACAGATTTTGGGCG No 17
ATCACAGATTTTGGGCGG No 18 TCACAGATTTTGGGCGGG No 19
ATTTTGGGCGGGCCAAAC No 20 AGATTTTGGGCGGGCCA No 21 ATCACAGATTTTGGGCGG
Yes * The bolded underlined base is the location of the L858R
mutant base.
Example 7
EGFR Mutation Detection in Paired Tissue and Plasma Samples
[0243] Paired FFPE tissue and plasma were obtained from 11 NSCLC
donors with informed consent (10 from Indivumed, Hamburg, Germany;
1 from Good Samaritan Hospital, Kearney, Nebr.). DNA from FFPE
tissue was analyzed by PCR followed by direct sequencing to
determine mutation status of the paired tissue/plasma donors. One
donor provided two plasma samples (856 and 3107) 1 year apart for
analysis. In addition, 6 normal plasma samples and 5 plasma samples
spiked with E746_A750del or L858R mutations were analyzed by the
methods described herein.
[0244] Results of direct sequencing of FFPE tissue and multiplex
fluorescent RF-PCR of plasma samples are presented in Table 3 along
with tumor size and overall plasma DNA concentration. Overall 3/11
(27%) NSCLC donors had mutation positive FFPE tissue, while the
remaining 8 were negative for the two mutations. Of the paired
NSCLC samples, 83.3% (10/12) of plasma samples demonstrated
identical mutation status to the matched FFPE tissue specimens. The
two E746_A750del positive plasma samples were drawn from the same
donor 1 year apart, thus 81.8% (9/11) unique donors had identical
mutation status between paired samples. Notably, although the
overall plasma DNA concentration had decreased by nearly half, the
latter plasma specimen had twice as many positive wells as the
specimen drawn 1 year previous (data not shown), suggesting the
circulating mutation concentration had increased in that time.
[0245] For the mutation spiked plasma samples, 100% of samples
spiked with 100, 200, and 300 pg of E746_A750del positive DNA
(H1650 cell line) and 100% of samples spiked with 100 and 300 pg of
L858R positive DNA (H1975 cell line) tested positive for their
respective mutation. Furthermore, 6 normal plasma specimens tested
negative for either mutation as expected.
TABLE-US-00009 TABLE 9 EGFR Mutation Detection in Paired Tissue and
Plasma Samples Tumor EGFR Mutation Detected Sample Diameter Plasma
DNA Tissue Plasma ID (cm) Conc. (ng/mL) PCR/Direct Seq RF-PCR NSCLC
856.sup.1 6.5.sup.2 15.6 E746_A750del E746_A750del 3107.sup.1
6.5.sup.2 8.1 E746_A750del E746_A750del 378 8.5 144 -- -- 401 5
20.6 -- -- 455 3.4 212 -- -- 477 3.5 54.0 -- -- 497 2.5 26.5 L858R
-- 516 5.7 11.1 -- -- 532 3.5 41.5 -- -- 563 1.9 265 L858R -- 631
2.9 128 -- -- 662 10.5 386 -- -- E746_A750del Spiked 200 pg N/A
23.3 N/A E746_A750del 300 pg N/A 9.9 N/A E746_A750del 100 pg N/A
7.6 N/A E746_A750del L858R spiked 300 pg N/A 8.0 N/A L858R 100 pg
N/A 6.4 N/A L858R Normal 4207 N/A 4.3 N/A -- 2011 N/A 5.8 N/A --
2258 N/A 8.7 N/A -- 3391 N/A 7.4 N/A -- 975 N/A 4.4 N/A -- 725 N/A
7.1 N/A -- .sup.1Sample IDs 856 and 3107 are from the same patient.
Sample ID 3107 was drawn 1 year after Sample ID 856. .sup.2Tumor
size measured 4.7 years prior to initial draw and 5.7 years prior
to second draw.
[0246] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0247] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising," "including," "containing," etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed.
[0248] Thus, it should be understood that although the present
invention has been specifically disclosed by preferred embodiments
and optional features, modification, improvement and variation of
the inventions embodied therein herein disclosed may be resorted to
by those skilled in the art, and that such modifications,
improvements and variations are considered to be within the scope
of this invention. The materials, methods, and examples provided
here are representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the
invention.
[0249] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0250] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0251] All publications, patent applications, patents, and other
references mentioned herein are expressly incorporated by reference
in their entirety, to the same extent as if each were incorporated
by reference individually. In case of conflict, the present
specification, including definitions, will control.
[0252] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
35120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1cccgtcgcta tcaaaacatc 20221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2atgtggagat gagcagggtc t 21321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3cttccacaat ggttgaacta g
21420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4catccatgtc cctacaaagg 20522DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5gagccattta tacagaaaga tg 22620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6gaaataccat ttgacccagc
20717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7tcacagattt tgggcgg 17820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8cctggtgtca ggaaaatgct 20919DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9cgagctaagc aggaggcgg
191020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10gtccatagtc gctggaggag 201119DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11cgagctaagc aggaggcgg 191221DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 12actttcagcc tgatagtctg g
211317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13atcacagatt ttgggcg 171421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14caagatcaca gattttgggc g 211525DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15atgtcaagat cacagatttt
gggcg 251619DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 16agatcacaga ttttgggcg
191718DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17atcacagatt ttgggcgg 181818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18tcacagattt tgggcggg 181918DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 19attttgggcg ggccaaac
182017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20agattttggg cgggcca 172118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21atcacagatt ttgggcgg 1822770DNAHomo sapiens 22ccacacggac
tttataacag gctttacaag cttgagattc ttttatctaa ataatcagtg 60tgattcgtgg
agcccaacag ctgcagggct gcgggggcgt cacagccccc agcaatatca
120gccttaggtg cggctccaca gccccagtgt ccctcacctt cggggtgcat
cgctggtaac 180atccacccag atcactgggc agcatgtggc accatctcac
aattgccagt taacgtcttc 240cttctctctc tgtcataggg actctggatc
ccagaaggtg agaaagttaa aattcccgtc 300gctatcaagg aattaagaga
agcaacatct ccgaaagcca acaaggaaat cctcgatgtg 360agtttctgct
ttgctgtgtg ggggtccatg gctctgaacc tcaggcccac cttttctcat
420gtctggcagc tgctctgctc tagaccctgc tcatctccac atcctaaatg
ttcactttct 480atgtctttcc ctttctagct ctagtgggta taactccctc
cccttagaga cagcactggc 540ctctcccatg ctggtatcca ccccaaaagg
ctggaaacag gcaattactg gcatctaccc 600agcactagtt tcttgacacg
catgatgagt gagtgctctt ggtgagcctg gagcatgggt 660attgtttttg
gtattttttg gatgaagaaa tggaggcata aagaaattgg ctgaccctta
720tatggctggg atagggttta agccccttgt tatttctgac tctgaaactt
77023755DNAHomo sapiens 23ccacacggac tttataacag gctttacaag
cttgagattc ttttatctaa ataatcagtg 60tgattcgtgg agcccaacag ctgcagggct
gcgggggcgt cacagccccc agcaatatca 120gccttaggtg cggctccaca
gccccagtgt ccctcacctt cggggtgcat cgctggtaac 180atccacccag
atcactgggc agcatgtggc accatctcac aattgccagt taacgtcttc
240cttctctctc tgtcataggg actctggatc ccagaaggtg agaaagttaa
aattcccgtc 300gctatcaaaa catctccgaa agccaacaag gaaatcctcg
atgtgagttt ctgctttgct 360gtgtgggggt ccatggctct gaacctcagg
cccacctttt ctcatgtctg gcagctgctc 420tgctctagac cctgctcatc
tccacatcct aaatgttcac tttctatgtc tttccctttc 480tagctctagt
gggtataact ccctcccctt agagacagca ctggcctctc ccatgctggt
540atccacccca aaaggctgga aacaggcaat tactggcatc tacccagcac
tagtttcttg 600acacgcatga tgagtgagtg ctcttggtga gcctggagca
tgggtattgt ttttggtatt 660ttttggatga agaaatggag gcataaagaa
attggctgac ccttatatgg ctgggatagg 720gtttaagccc cttgttattt
ctgactctga aactt 755241562DNAArtificial SequenceDescription of
Artificial Sequence Synthetic construct 24ccagctccgt tcagagtgaa
ccatgcagtg gaatggtaag tggcattata agccccagtg 60atcttccaga tagccctgga
caaaccatgc caccaagcag aagtaaaaca cctccaccac 120ctcctcaaac
agctcaaacc aagcgagaag tacctaaaaa taaagcacct actgctgaaa
180agagagagag tggacctaag caagctgcag taaatgctgc agttcagagg
gtccaggttc 240ttccagatgc tgatacttta ttacattttg ccacggaaag
tactccagat ggattttctt 300gttcatccag cctgagtgct ctgagcctcg
atgagccatt tatacagaaa gatgtggaat 360taagaataat gtgcatgtgt
ctttatagca gcatgattta tactcatttg ggtatatacc 420cagtaatggg
atggctgggt caaatggtat ttctagttct agatccctga ggaatcgcca
480cactgacttc cacaatggtt gaactagttt acagtcccac caagaaaatg
tggcacatat 540acaccatgga atactatgca gccataaaaa atgatgagtt
catatccttt gtagggacat 600ggatgaaatt ggaaaccatc attctcagta
aactatcgca agaacaaaaa accaaacacc 660gcatattctc acttataggt
gggaattgaa caatgagatc acatggacac aggaagggga 720atatcacact
ctggggactg tggtggggtc gggggagggg ggagggatag cattgggaga
780tatacctaat gctagatgac acattagtgg gtgcagcgca gcatggcaca
tgtatacata 840tgtaactaac ctgcacaatg tgcacatgta ccctaaaact
tagagtataa taaaaaaaaa 900aaaaaaaaaa ataacaataa atgagataaa
atctaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agaataatgc
ctccagttca 1140ggaaaatgac aatgggaatg aaacagaatc agagcagcct
aaagaatcaa atgaaaacca 1200agagaaagag gcagaaaaaa ctattgattc
tgaaaaggac ctattagatg attcagatga 1260tgatgatatt gaaatactag
aagaatgtat tatttctgcc atgccaacaa agtcatcacg 1320taaagcaaaa
aagccagccc agactgcttc aaaattacct ccacctgtgg caaggaaacc
1380aagtcagctg cctgtgtaca aacttctacc atcacaaaac aggttgcaac
cccaaaagca 1440tgttagtttt acaccggggg atgatatgcc acgggtgtat
tgtgttgaag ggacacctat 1500aaacttttcc acagctacat ctctaagtga
tctaacaatc gaatcccctc caaatgagtt 1560ag 1562251562DNAArtificial
SequenceDescription of Artificial Sequence Synthetic construct
25ccagctccgt tcagagtgaa ccatgcagtg gaatggtaag tggcattata agccccagtg
60atcttccaga tagccctgga caaaccatgc caccaagcag aagtaaaaca cctccaccac
120ctcctcaaac agctcaaacc aagcgagaag tacctaaaaa taaagcacct
actgctgaaa 180agagagagag tggacctaag caagctgcag taaatgctgc
agttcagagg gtccaggttc 240ttccagatgc tgatacttta ttacattttg
ccacggaaag tactccagat ggattttctt 300gttcatccag cctgagtgct
ctgagcctcg atgagccatt tatacagaaa gatgtggaat 360taagaataat
gtgcatgtgt ctttatagca gcatgattta tactcatttg ggtatatacc
420cagtaatggg atggctgggt caaatggtat ttctagttct agatccctga
ggaatcgcca 480cactgacttc cacaatggtt gaactagttt acagtcccac
caagaaaatg tggcacatat 540acaccatgga atactatgca gccataaaaa
atgatgagtt catatccttt gtagggacat 600ggatgaaatt ggaaaccatc
attctcagta aactatcgca agaacaaaaa accaaacacc 660gcatattctc
acttataggt gggaattgaa caatgagatc acatggacac aggaagggga
720atatcacact ctggggactg tggtggggtc gggggagggg ggagggatag
cattgggaga 780tatacctaat gctagatgac acattagtgg gtgcagcgca
gcatggcaca tgtatacata 840tgtaactaac ctgcacaatg tgcacatgta
ccctaaaact tagagtataa taaaaaaaaa 900aaaaaaaaaa ataacaataa
atgagataaa atctaaaaaa aaaaaaaaaa aaaaaaaaaa 960aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1020aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1080aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
agaataatgc ctccagttca 1140ggaaaatgac aatgggaatg aaacagaatc
agagcagcct aaagaatcaa atgaaaacca 1200agagaaagag gcagaaaaaa
ctattgattc tgaaaaggac ctattagatg attcagatga 1260tgatgatatt
gaaatactag aagaatgtat tatttctgcc atgccaacaa agtcatcacg
1320taaagcaaaa aagccagccc agactgcttc aaaattacct ccacctgtgg
caaggaaacc 1380aagtcagctg cctgtgtaca aacttctacc atcacaaaac
aggttgcaac cccaaaagca 1440tgttagtttt acaccggggg atgatatgcc
acgggtgtat tgtgttgaag ggacacctat 1500aaacttttcc acagctacat
ctctaagtga tctaacaatc gaatcccctc caaatgagtt 1560ag 15622639DNAHomo
sapiens 26cagcatgtca agatcacaga ttttgggctg gccaaactg 392739DNAHomo
sapiens 27cagcatgtca agatcacaga ttttgggcgg gccaaactg 3928524DNAHomo
sapiens 28tgaccctgaa ttcggatgca gagcttcttc ccatgatgat ctgtccctca
cagcagggtc 60ttctctgttt cagggcatga actacttgga ggaccgtcgc ttggtgcacc
gcgacctggc 120agccaggaac gtactggtga aaacaccgca gcatgtcaag
atcacagatt ttgggctggc 180caaactgctg ggtgcggaag agaaagaata
ccatgcagaa ggaggcaaag taaggaggtg 240gctttaggtc agccagcatt
ttcctgacac cagggaccag gctgccttcc cactagctgt 300attgtttaac
acatgcaggg gaggatgctc tccagacatt ctgggtgagc tcgcagcagc
360tgctgctggc agctgggtcc agccagggtc tcctggtagt gtgagccaga
gctctgaggt 420ttcactctgg cctgctgggc tcctagcagc caccaaccca
tgatgctggg ccctgaaaac 480acacgcagac ctggatgagt gaggccactg
ggcacaacca gggc 52429413DNAHomo sapiens 29tgaccctgaa ttcggatgca
gagcttcttc ccatgatgat ctgtccctca cagcagggtc 60ttctctgttt cagggcatga
actacttgga ggaccgtcgc ttggtgcacc gcgacctggc 120agccaggaac
gtactggtga aaacaccgca gcatgtcaag atcacagatt ttgggcgggc
180caaactgctg ggtgcggaag agaaagaata ccatgcagaa ggaggcaaag
taaggaggtg 240gctttaggtc agccagcatt ttcctgacac cagggaccag
gctgccttcc cactagctgt 300attgtttaac acatgcaggg gaggatgctc
tccagacatt ctgggtgagc tcgcagcagc 360tgctgctggc agctgggtcc
agccagggtc tcctggtagt gtgagccaga gct 41330316DNAHomo sapiens
30tgtcgccctg gaccctggga caccgcctcc tgagattaaa gcgagagcca gggcgggccg
60ggccgagtag gcgcgagcta agcaggaggc ggaggcggag gcggagggcg aggggcgggg
120agcgccgcct ggagcgcggc aggtcatatt gaacattcca gatacctatc
attactcgat 180gctgttgata acagcaagat ggctttgaac tcagggtcac
caccagctat tggaccttac 240tatgaaaacc atggatacca accggaaaac
ccctatcccg cacagcccac tgtggtcccc 300actgtctacg aggtgc
31631364DNAHomo sapiens 31ccaaaagcaa gacaaatgac tcacagagaa
aaaagatggc agaaccaagg gcaactaaag 60ccgtcaggtt ctgaacagct ggtagatggg
ctggcttact gaaggacatg attcagactg 120tcccggaccc agcagctcat
atcaaggaag ccttatcagt tgtgagtgag gaccagtcgt 180tgtttgagtg
tgcctacgga acgccacacc tggctaagac agagatgacc gcgtcctcct
240ccagcgacta tggacagact tccaagatga gcccacgcgt ccctcagcag
gattggctgt 300ctcaaccccc agccagggtc accatcaaaa tggaatgtaa
ccctagccag gtgaatggct 360caag 36432360DNAArtificial
SequenceDescription of Artificial Sequence Synthetic construct
32tgtcgccctg gaccctggga caccgcctcc tgagattaaa gcgagagcca gggcgggccg
60ggccgagtag gcgcgagcta agcaggaggc ggaggcggag gcggagggcg aggggcgggg
120agcgccgcct ggagcgcggc aggaagcctt atcagttgtg agtgaggacc
agtcgttgtt 180tgagtgtgcc tacggaacgc cacacctggc taagacagag
atgaccgcgt cctcctccag 240cgactatgga cagacttcca agatgagccc
acgcgtccct cagcaggatt ggctgtctca 300acccccagcc agggtcacca
tcaaaatgga atgtaaccct agccaggtga atggctcaag 36033316DNAHomo sapiens
33tgtcgccctg gaccctggga caccgcctcc tgagattaaa gcgagagcca gggcgggccg
60ggccgagtag gcgcgagcta agcaggaggc ggaggcggag gcggagggcg aggggcgggg
120agcgccgcct ggagcgcggc aggtcatatt gaacattcca gatacctatc
attactcgat 180gctgttgata acagcaagat ggctttgaac tcagggtcac
caccagctat tggaccttac 240tatgaaaacc atggatacca accggaaaac
ccctatcccg cacagcccac tgtggtcccc 300actgtctacg aggtgc
31634355DNAHomo sapiens 34tgcgaagagc agcagcatgg atggatttta
tgaccagcaa gtgccttaca tggtcaccaa 60tagtcagcgt gggagaaatt gtaacgagaa
accaacaaat gtcaggaaaa gaaaattcat 120taacagagat ctggctcatg
attcagaaga actctttcaa gatctaagtc aattacagga 180aacatggctt
gcagaagctc aggtacctga caatgatgag cagtttgtac cagactatca
240ggctgaaagt ttggcttttc atggcctgcc actgaaaatc aagaaagaac
cccacagtcc 300atgttcagaa atcagctctg cctgcagtca agaacagccc
tttaaattca gctat 35535300DNAArtificial SequenceDescription of
Artificial Sequence Synthetic construct 35tgtcgccctg gaccctggga
caccgcctcc tgagattaaa gcgagagcca gggcgggccg 60ggccgagtag gcgcgagcta
agcaggaggc ggaggcggag gcggagggcg aggggcgggg 120agcgccgcct
ggagcgcggc agctcaggta cctgacaatg atgagcagtt tgtaccagac
180tatcaggctg aaagtttggc ttttcatggc ctgccactga aaatcaagaa
agaaccccac 240agtccatgtt cagaaatcag ctctgcctgc agtcaagaac
agccctttaa attcagctat 300
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